Prophylaxis of colorectal and gastrointestinal cancer

ABSTRACT

The present disclosure provides methods and compositions useful for preventing gastrointestinal and/or colorectal cancer in animals, including humans, having pre-cancerous adenomatous polyps. The present disclosure provides compositions comprising anti-PG antibodies suitable for use in the methods of the disclosure. The present disclosure also provides methods and compositions useful for monitoring the efficacy of anti-PG treatment in subjects with pre-cancerous polyps.

1. REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) ofprovisional application No. 61/317,245, filed Mar. 24, 2010, the contentof which is incorporated by reference in its entirety.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith.

3. FIELD OF THE INVENTION

The present disclosure is directed to, among other things, methods ofpreventing colorectal and/or gastrointestinal cancer in subjectspredisposed to develop adenomatous polyps by administering to thesubject a composition comprising an antibody specific for progastrin.

4. BACKGROUND

Cancer of the gastrointestinal tract, including colorectal cancer(“CRC”), affects hundreds of thousands of individuals every year andtens of thousands of CRC-related deaths occur every year in the UnitedStates alone. See, Rustgi, 2010, “The genetics of hereditary coloncancer,” Genes & Development 21:2525-2538. CRC can arise in differentways, one of which is the transformation of adenomatous polyps intomalignant tumors. Adenomatous polyposis may be inherited, as is the casefor individuals with familial adenomatous polyposis (“FAP”), or it maybe sporadic. Individuals with FAP or sporadic adenomatous polyposiscarry mutations of the Adenomatous Polyposis Coli (“APC”) tumorsuppressor gene which are associated with the formation of adenomatouspolyps in the small intestine, colon and/or rectum. These polyps in turncan develop into colorectal and gastrointestinal cancer. In the case ofsporadic adenomatous polyposis, a non-hereditary condition thatunderlies many instances of CRC, the APC gene is mutated in somaticcells. Individuals with sporadic adenomatous polyposis develop benignpolyps, a subset of which may subsequently transform into malignantcarcinomas.

FAP accounts for around 1% of total CRC cases and affects one in 13,000births. Id. Mutation of APC in FAP patients is associated with theformation of hundreds to thousands of small adenomatous polypsthroughout the colon. Progression of polyps to malignancy is virtuallyinevitable. On average, without prophylactic treatment, individuals withFAP develop CRC by age 39. Prophylactic treatment is the standard ofcare and involves radical surgery, including the removal of the colon,or of both the colon and the rectum, generally before the age of 25.While prophylaxis is preferable to no treatment, surgical resection ofthe colon (colectomy) in young patients severely impairs quality oflife. In addition, surgical resection alone may be inadequate to keeppatients cancer-free: patients who have colectomies have a high risk ofdeveloping polyps and cancer in the upper gastrointestinal tract. Thereis a serious need for effective prophylactic treatments, especiallynon-surgical treatments, that extend cancer-free life for individualswith FAP and individuals with sporadic adenomatous polyposis.

5. SUMMARY

The present disclosure provides methods and compositions useful forpreventing gastrointestinal cancer, including CRC, in animals, includinghumans, predisposed to developing adenomatous polyps. As describedbelow, the present application sets forth treatment regimens believed tobind progastrin (“PG”), with the apparent ability to neutralize PG'sbiological activity, which are useful in subjects who have an increasedlikelihood of developing, but have not yet developed, CRC or cancer inthe upper gastrointestinal tract. The various inventions described inthe application are based in part on the applicants' discovery thatanti-PG antibodies prevent the development of gastrointestinal tumors ina mouse model of FAP. While not intending to be bound by any theory ofoperation, binding PG and interfering with its interaction with otherproteins in the body is thought to prevent adenomatous polyps fromdeveloping into malignant tumors.

Accordingly, in one aspect, the present disclosure provides methods ofpreventing gastrointestinal cancer, including CRC, in subjectspredisposed to developing adenomatous polyps by administering acomposition comprising an anti-PG antibody. Generally, the methodscomprise administering to a subject in need thereof an effective amountof an anti-PG antibody. Anti-PG antibodies, and compositions thereof,can be administered according to regimens known in the art forantibody-based therapy, at an effective dosage, i.e., an amounteffective to prevent or delay gastrointestinal cancer, including CRC, ina subject.

Suitable subjects for prophylactic anti-PG treatment are thosepredisposed to developing adenomatous polyps, including subjects with afamily history of CRC, individuals with FAP, and those in whomadenomatous polyps have previously been found and/or removed. Typically,suitable subjects have one or more mutations in the APC gene, leading toFAP or sporadic adenomatous polyposis. Suitable subjects also includeindividuals who have previously had a colectomy and are at increasedrisk of developing polyps and cancer in the upper gastrointestinaltract.

Anti-PG antibodies of the present disclosure include antibodies capableof binding PG. Any antibody capable of binding PG may be used in themethods of the present disclosure, including, but not limited to,polyclonal and monoclonal anti-PG antibodies. Preferably, the anti-PGantibody is specific to the PG of the species being treated. Forexample, an anti-human PG (anti-hPG) antibody is administered to a humansubject. Suitable anti-PG antibodies can range in binding affinity fromat least about 5000 nM to at least about 0.001 nM, or higher, or anyvalue in between.

Anti-PG antibodies described herein can be used in combination with, oradjunctive to, other treatments to prevent or delay gastrointestinalcancer, including CRC. Non-limiting examples of other treatments includesurgical resection, chemotherapy, antibody therapy, radiation therapy,and treatment with a second agent as described herein. Anti-PGantibodies can be administered concurrently with, or at a time before orafter, another treatment.

Compositions suitable for use in the methods of the present disclosuremay comprise, in addition to an anti-PG antibody, a pharmaceuticallyacceptable carrier, excipient, and/or diluent. The compositions can beformulated for various routes of administration as described herein,comprising carriers, excipients, and/or diluents suitable for the chosenroute. For treatment in humans and animals, compositions comprisinganti-PG antibodies can be administered using any suitable route ofadministration, such as injection and other routes of administrationknown in the art for antibody-based clinical products. For treatmentpurposes, compositions can be packaged in unit doses for ease of use.

As shown herein, patients with multiple adenomatous polyps have elevatedserum PG levels, whereas patients in whom polyps have been removed havelow or undetectable serum PG levels. This discovery provides powerfulnew tools to diagnose and monitor the course of sporadic or familialadenomatous polyposis and its treatment.

Accordingly, in another aspect, the present disclosure provides methodsof monitoring the efficacy of anti-PG treatment in an individualpredisposed to developing adenomatous polyps. Generally, the methodscomprise measuring a concentration, or level, of PG in a blood (serum,plasma, or whole blood) sample from the individual receiving anti-PGtherapy, during or after a course of anti-PG therapy, and comparing themeasured PG level to a baseline level of PG (e.g., a PG level in theindividual at the start of treatment), wherein a measured PG level belowthat of the baseline level is indicative of treatment efficacy and ameasured PG level above that of the baseline level is indicative of alack of efficacy. In some embodiments, the method further includesassessing the number and sizes of polyps in the subject by, for example,endoscopy.

In yet another aspect, the present disclosure provides methods forselecting individuals, in whom endoscopy or anti-PG treatment isindicated. The methods are intended to be carried out in individualspredisposed to developing adenomatous polyps. Generally, the method iscarried out by measuring the level of PG in a blood sample from theindividual, and comparing the measured level of PG to a baseline level,where a measured PG level higher than the baseline level indicates aneed for endoscopy. In some embodiments, a PG level above the baselineindicates a need for anti-PG treatment. The baseline can be obtainedfrom one or more samples from the individual at an earlier point intime, or can be based upon PG levels measured in a population havingcharacteristics similar to the individual.

6. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides amino acid sequences of human preprogastrin (SEQ IDNO:100), where the signal peptide sequence is underlined, mature humanprogastrin (SEQ ID NO:20) and certain products of progastrin processing,including G34 (SEQ ID NO:102), G34-Gly (SEQ ID NO:103), G17 (SEQ IDNO:104), G17-Gly (SEQ ID NO:105) and CTFP (SEQ ID NO:106).

FIG. 2. provides polynucleotide and amino acid sequences of variablelight and variable heavy chains of certain exemplary murine anti-hPGmonoclonal antibodies. In each case, the three CDRs are shown inbolded-underlined text. Specifically:

FIG. 2A provides the polypeptide sequence of the V_(H) chain of murineanti-hPG MAb3 (SEQ ID NO:12) and a polynucleotide sequence encoding it(SEQ ID NO:16);

FIG. 2B provides the polypeptide sequence of the V_(L) chain of murineanti-hPG MAb3 (SEQ ID NO:13) and a polynucleotide sequence encoding it(SEQ ID NO:17);

FIG. 2C provides the polypeptide sequence of the V_(H) chain of murineanti-hPG MAb4 (SEQ ID NO:14) and a polynucleotide sequence encoding it(SEQ ID NO:18);

FIG. 2D provides the polypeptide sequence of the V_(L) chain of murineanti-hPG MAb4 (SEQ ID NO:15) and a polynucleotide sequence encoding it(SEQ ID NO:19);

FIG. 2E provides the polypeptide sequence of the V_(H) chain of murineanti-hPG MAb8 (SEQ ID NO:59) and a polynucleotide sequence encoding it(SEQ ID NO:67);

FIG. 2F provides the polypeptide sequence of the V_(L) chain of murineanti-hPG MAb8 (SEQ ID NO:63) and a polynucleotide sequence encoding it(SEQ ID NO:71);

FIG. 2G provides the polypeptide sequence of the V_(H) chain of murineanti-hPG MAb13 (SEQ ID NO:60) and a polynucleotide sequence encoding it(SEQ ID NO:68);

FIG. 2H provides the polypeptide sequence of the V_(L) chain of murineanti-hPG MAb13 (SEQ ID NO:64) and a polynucleotide sequence encoding it(SEQ ID NO:72);

FIG. 2I provides the polypeptide sequence of the V_(H) chain of murineanti-hPG MAb16 (SEQ ID NO:61) and a polynucleotide sequence encoding it(SEQ ID NO:69);

FIG. 2J provides the polypeptide sequence of the V_(L) chain of murineanti-hPG MAb16 (SEQ ID NO:65) and a polynucleotide sequence encoding it(SEQ ID NO:73);

FIG. 2K provides the polypeptide sequence of the V_(H) chain of murineanti-hPG MAb19 (SEQ ID NO:62) and a polynucleotide sequence encoding it(SEQ ID NO:70); and

FIG. 2L provides the polypeptide sequence of the V_(L) chain of murineanti-hPG MAb19 (SEQ ID NO:66) and a polynucleotide sequence encoding it(SEQ ID NO:74).

FIG. 3 provides projected polypeptide sequences for humanized variableheavy and light chains of selected anti-hPG monoclonal antibodiesdescribed herein. In each case, the three CDRs are shown inbolded-underlined text. Specifically:

FIG. 3A provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb3 (SEQ ID NO:21);

FIG. 3B provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb3 (SEQ ID NO:22);

FIG. 3C provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb4 (SEQ ID NO:23);

FIG. 3D provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb4 (SEQ ID NO:24);

FIG. 3E provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb8(a) (SEQ ID NO:75);

FIG. 3F provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb8(a) (SEQ ID NO:76);

FIG. 3G provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb8(b) (SEQ ID NO:77);

FIG. 3H provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb8(b) (SEQ ID NO:78);

FIG. 3I provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb8(c) (SEQ ID NO:79);

FIG. 3J provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb8(c) (SEQ ID NO:76);

FIG. 3K provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb13(a) (SEQ ID NO:80);

FIG. 3L provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb13(a) (SEQ ID NO:81);

FIG. 3M provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb13(b) (SEQ ID NO:82);

FIG. 3N provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb13(b) (SEQ ID NO:83);

FIG. 3O provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb16(a) (SEQ ID NO:84);

FIG. 3P provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb16(a) (SEQ ID NO:85);

FIG. 3Q provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb16(b) (SEQ ID NO:86);

FIG. 3R provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb16(b) (SEQ ID NO:87);

FIG. 3S provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb16(c) (SEQ ID NO:88);

FIG. 3T provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb16(c) (SEQ ID NO:89);

FIG. 3U provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb19(a) (SEQ ID NO:90);

FIG. 3V provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb19(a) (SEQ ID NO:91);

FIG. 3W provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb19(b) (SEQ ID NO:92);

FIG. 3X provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb19(b) (SEQ ID NO:93);

FIG. 3Y provides the projected amino acid sequence of the V_(H) chain ofhumanized MAb19(c) (SEQ ID NO:94); and

FIG. 3Z provides the projected amino acid sequence of the V_(L) chain ofhumanized MAb19(c) (SEQ ID NO:95).

7. DETAILED DESCRIPTION 7.1 Cancer in Familial and Sporadic AdenomatousPolyposis

Familial Adenomatous Polyposis (FAP) is a rare hereditary conditionassociated with a germinal mutation on one allele of the APC gene.Numerous mutations of the APC gene have been mapped in subjects withFAP, many of the mutations resulting in a truncated protein. See, e.g.,Rustgi, 2010, “The genetics of hereditary colon cancer,” Genes &Development 21:2525-2538; Groves et al., 2002, “Duodenal cancer inpatients with familial adenomatous polyposis (FAP): results of a 10 yearprospective study,” Gut 50:636-641. These mutations in APC areassociated with FAP of varying severity.

FAP is characterized by the appearance of multiple adenomas (polyps) inthe intestine and colon of affected individuals, at a very young age. Asubset of these polyps transform into colorectal cancer (CRC), andFAP-derived CRC cases represent approximately 1% of total CRC cases.Where there is a family history of CRC, individuals typically undergogenetic testing at a very early age to detect the presence of a mutationin the APC gene. Classical follow-up in individuals found to have suchmutations begins with colonoscopy, polyp resection where polyps arefound, and if polyps are too numerous to remove endoscopically, partialor complete resection of the colon (colectomy). Most individuals withFAP will have undergone colectomy by the age of 25.

A large percentage of sporadic adenomatous polyps also harbor mutationsin the APC gene. See, Rutsgi, 2010, “The genetics of hereditary coloncancer,” Genes & Development 21:2525-2538. In the absence of any familyhistory of CRC, individuals presenting with symptoms such as rectalbleeding are typically examined by colonoscopy. Individuals found tohave large numbers of polyps, or in whom polyps recur after resection,will typically also be tested genetically. If a mutation in the APC geneis found, colectomy is the recommended treatment.

Even after colectomy, individuals predisposed to developing adenomatouspolyps have an increased risk of developing adenomatous polyps in theunresected portion of their gastrointestinal tracts. Such individualsare regularly followed by endoscopy and assessed for adenomatosis in theupper gastrointestinal tract. Individuals are staged according to theSpigelman classification, which relies on four parameters to evaluatethe degree or severity of adenomatosis: number of polyps, size ofpolyps, histology of polyps, and degree of polyp dysplasia (disorderedgrowth). See, Spigelman et al., 1989, “Upper gastrointestinal cancer inpatients with Familial Adenomatous Polyposis,” Lancet 2:783-785. TheSpigelman classification categorizes individuals into one of five stagesfor duodenal polyposis, Stage 0 to IV, based on the four parameters, asshown in Table 1:

TABLE 1 Spigelman Classification Number of Polyp size Points polyps (mm)Histology Dysplasia 1 1-4  1-4  Tubular Mild 2 5-20 5-10 TubulovillousModerate 3 >20 >10 Villous Severe Stage I: 1-4 points; Stage II: 5-6points; Stage III: 7-8 points; Stage IV: 9-12 points.A ten-year study of 114 individuals with FAP revealed that SpigelmanStage IV patients had a 36.4% risk of developing duodenal cancer, ascompared to a 0% to 2.4% risk for patients classified in Stages 0 toIII. See, Groves et al., 2002, “Duodenal cancer in patients withfamilial adenomatous polyposis (FAP): results of a 10 year prospectivestudy,” Gut 50:636-641.

It has previously been shown that approximately 70% of patients with CRChave elevated levels of PG. As shown in the Examples below, Applicantshave now discovered that blood levels of PG can be elevated inindividuals with FAP who have not yet developed CRC, as well as in about20% of patients exhibiting sporadic adenomatous polyposis. While notintending to be bound by any theory of operation, PG is thought to bepart of the mechanism by which polyps transition to malignant tumors.Binding of PG by anti-PG antibodies is thought to interfere with thistransition, as demonstrated in the mouse model of FAP, APCΔ14.Prophylactic treatment with anti-PG antibodies presents the possibilityof avoiding or delaying major surgery, significantly increasing qualityof life.

7.2 Methods of Prophylaxis

The present disclosure provides methods of preventing gastrointestinalcancer, including CRC, in patients predisposed to developing adenomatouspolyps. Generally, the methods comprise administering to such patientsan amount of one or more anti-PG antibody(ies) effective to provide atherapeutic benefit. Anti-PG antibodies generally, and specific anti-PGantibodies useful in the methods, are described in detail in a latersection.

The “subject” or “patient” for prophylaxis is preferably a mammal suchas a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or aprimate (e.g., monkey or human). The anti-PG antibody administeredshould be specific for the species of animal being treated. Fortreatment of human subjects, the anti-PG antibody(ies) shouldspecifically bind human progastrin (referred to herein as “anti-hPGantibodies,” described in more detail below).

The subject or patient can be a human, such as an adult patient or apediatric patient. Suitable subjects are individuals who are predisposedto developing adenomatous polyps and, as a result, have an increasedlikelihood of developing CRC or gastrointestinal cancer, includingindividuals with a family history of CRC, individuals in whomadenomatous polyps are or have been detected or removed, individualswith FAP, individuals who have had a colectomy to remove polyps, andindividuals with mutation(s) in the APC gene.

Anti-PG treatment can be administered in combination with, or adjunctiveto, one or more other treatments to prevent or delay gastrointestinalcancer, including CRC. Other treatments include, without limitation,chemotherapeutic treatment, radiation, surgical resection, antibodytherapy, and treatment with a second agent, as described herein.Combination treatment as provided herein involves the administration ofat least two treatments to a patient, the first of which is anti-PGtreatment with at least one anti-PG antibody, and the second of which istreatment with a therapeutic or prophylactic agent or procedure.

Anti-PG treatment can be combined with surgical procedures, such assurgical resection. Anti-PG antibodies can be administered to subjectsfound to have, or predisposed to develop, pre-cancerous polyps, such asindividuals with familial adenomatous polyposis, in combination withsurgical resection of the affected portion(s) of the gastrointestinaltract. Anti-PG treatment can be initiated before, concurrently with, orafter surgical resection.

Anti-PG treatment can also be combined with radiation therapy. Radiationtherapy is the use of high-energy radiation from x-rays, gamma rays,neutrons, protons, and other sources to kill cancer cells and shrinktumors. Radiation may come from a machine outside the body(external-beam radiation therapy), or it may come from radioactivematerial placed in the body near cancer cells (internal radiationtherapy, or brachytherapy). Systemic radiation therapy uses aradioactive substance, such as a radiolabeled monoclonal antibody, thattravels in the blood to tissues throughout the body. Radiation therapymay also be called irradiation and radiotherapy. Other radiationtherapies include three-dimensional conformal radiation therapy (3D-CRT)and intensity modulated radiation therapy (IMRT). Other radiationtherapies are also possible.

Where anti-PG antibody treatment is combined with a second agent, thesecond agent can be a chemotherapeutic agent. Chemotherapy is the use ofsmall molecule drugs that kill (cytotoxic or cytocidal) or prevent thegrowth (cytostatic) of cancer cells. Chemotherapeutic agents include,but are not limited to, toxins, also referred to as cytotoxins orcytotoxic agents, which includes any agent that is detrimental to theviability of cells, agents, and liposomes or other vesicles containingchemotherapeutic compounds. Examples of suitable chemotherapeutic agentsinclude but are not limited to 1-dehydrotestosterone, 5-fluorouracildecarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin,aldesleukin, alkylating agents, allopurinol sodium, altretamine,amifostine, anastrozole, anthramycin (AMC), anti-mitotic agents,cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase,BCG live (intravesical), betamethasone sodium phosphate andbetamethasone acetate, bicalutamide, bleomycin sulfate, busulfan,calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine(CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine,Colchicin, conjugated estrogens, Cyclophosphamide, Cyclothosphamide,Cytarabine, Cytarabine, cytochalasin B, Cytoxan, Dacarbazine,Dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCL,daunorucbicin citrate, denileukin diftitox, Dexrazoxane,Dibromomannitol, dihydroxy anthracin dione, Docetaxel, dolasetronmesylate, doxorubicin HCL, dronabinol, E. coli L-asparaginase, emetine,epoetin-α, Erwinia L-asparaginase, esterified estrogens, estradiol,estramustine phosphate sodium, ethidium bromide, ethinyl estradiol,etidronate, etoposide citrororum factor, etoposide phosphate,filgrastim, floxuridine, fluconazole, fludarabine phosphate,fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids,goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea,idarubicin HCL, ifosfamide, interferon α-2b, irinotecan HCL, letrozole,leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine,lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesteroneacetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna,methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane,mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL,oxaliplatin, paclitaxel, pamidronate disodium, pentostatin, pilocarpineHCL, plimycin, polifeprosan 20 with carmustine implant, porfimer sodium,procaine, procarbazine HCL, propranolol, rituximab, sargramostim,streptozotocin, tamoxifen, taxol, tegafur, teniposide, tenoposide,testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa,topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin,vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

Anti-PG antibodies can also be administered with a combination ofchemotherapeutic agents. Exemplary combinations of chemotherapeuticagents include 5-fluorouracil (5FU) in combination with leucovorin(folinic acid or LV); capecitabine, in combination with uracil (UFT) andleucovorin; tegafur in combination with uracil (UFT) and leucovorin;oxaliplatin in combination with 5FU, or in combination withcapecitabine; irinotecan in combination with capecitabine, mitomycin Cin combination with 5FU, irinotecan or capecitabine. Other combinationsof chemotherapeutic agents disclosed herein is also possible.

Standard dosing regimens for chemotherapeutic agents used for patientssuffering from CRC may be used in the methods of the present disclosure.As is known in the relevant art, chemotherapy regimes for colorectalcancer using combinations of different chemotherapeutic agents have beenstandardized in clinical trials. Such regimes are often known byacronyms and include 5FU Mayo, 5FU Roswell Park, LVFU2, FOLFOX, FOLFOX4,FOLFOX6, bFOL, FUFOX, FOLFIRI, IFL, XELOX, CAPDX, XELIRI, CAPIRI,FOLFOXIRI. See, e.g., Chau, I. et al., 2009, Br. J., Cancer 100:1704-19,and Field, K. et al., 2007, World J. Gastroenterol. 13:3806-15, both ofwhich are incorporated by reference.

Anti-PG antibodies can also be used in combination with otherantibodies, including but not limited to, monoclonal antibodies thatdirectly or indirectly kill, slow or stop the growth of cancer cells.Such antibodies can function through a variety of distinct mechanisms.For example, certain antibodies can mark cancer cells for attack by thepatient's immune system via antibody-dependent cell-mediatedcytotoxicity (ADCC) or other mechanisms. It is believed that rituximab(Rituxan®), which binds the CD20 antigen found on B cells, andedrecolomab, which binds the 17-1A antigen, function this way. Otherantibodies bind to and alter or inhibit the function of antigens thatcancer cells require for survival and/or growth. A number of antibodiesare believed to function this way, including, for example, cetuximab(Erbitux®) and panitumumab (Vectibix®), each of which binds to the EGFreceptor (EGFR); and bevacizumab (Avastin®), which binds to the growthfactor VEGF. Other mechanisms are also possible, and particularantibodies may be able to work via one or more mechanisms of action. Yetother antibodies can be conjugated to radioactive or chemotoxic moietiesand target them to cancer cells which preferentially express antigensspecifically recognized by the antibodies.

Anti-PG antibodies can also be administered in combination withnon-steroidal anti-inflammatory drugs (“NSAIDs”). For example,celecoxib, or4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide,is an NSAID that has been shown to reduce adenomatous polyps in FAPpatients.

The anti-PG antibody and a second agent can be administeredsimultaneously, successively, or separately. As used herein, the anti-PGantibody and the second agent are said to be administered successivelyif they are administered to the patient on the same day, for exampleduring the same patient visit. Successive administration can occur 1, 2,3, 4, 5, 6, 7 or 8 hours apart. In contrast, the anti-PG antibody andthe second agent are said to be administered separately if they areadministered to the patient on different days, for example, the anti-PGantibody and the second therapeutic agent can be administered at a1-day, 2-day or 3-day, one-week, 2-week or monthly intervals. In themethods of the present disclosure, administration of the anti-PGantibody of the disclosure can precede or follow administration of thesecond agent.

As a non-limiting example, the anti-PG antibody and second agent can beadministered concurrently for a period of time, followed by a secondperiod of time in which the administration of anti-PG antibody and thesecond agent are alternated.

7.3 Pharmaceutical Compositions And Kits

Anti-PG antibodies useful in the methods of the present disclosure canbe formulated in compositions. Optionally, the compositions can compriseone or more additional agent(s), such as the second agents describedabove. The compositions will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administering it to anindividual).

Anti-PG antibodies can be administered to an individual by a variety ofroutes such as orally, transdermally, subcutaneously, intranasally,intravenously, intramuscularly, intraocularly, topically, intrathecallyand intracerebroventricularly. The most suitable route foradministration in any given case will depend on the particular antibody,the subject, and the nature and severity of the disease and the physicalcondition of the subject. Antibodies can be formulated as an aqueoussolution and administered by subcutaneous injection. Pharmaceuticallyacceptable carriers for use in the disclosure can take a wide variety offorms depending, e.g., on the condition to be treated or route ofadministration.

Pharmaceutical compositions can be conveniently presented in unit doseforms containing a predetermined amount of an anti-PG antibody per dose.Such a unit can contain for example 5 mg to 5 g, for example 10 mg to 1g, or 20 to 50 mg of anti-PG antibody per unit dose. Pharmaceuticalcompositions can comprise anti-PG antibodies capable of binding morethan one PG epitope. Alternatively, pharmaceutical compositions maycomprise a combination of anti-PG antibodies, each capable of binding adifferent PG epitope.

Pharmaceutical compositions of the disclosure can be prepared forstorage as lyophilized formulations or aqueous solutions by mixing theantibody having the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, e.g., Remington's Pharmaceutical Sciences,16th edition (Osol, ed. 1980). Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can be present at concentration rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives can be added to retard microbial growth, and can be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present disclosure include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, and iodide),hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; and polysaccharides such as dextran.Stabilizers can be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188,etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/ml to about 1.0 mg/ml, for example about 0.07 mg/ml toabout 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

Anti-PG antibodies can be administered singly, as mixtures of one ormore anti-PG antibodies, in mixture or combination with other agentsuseful in preventing CRC, or adjunctive to therapy for CRC. Examples ofsuitable combination and adjunctive therapies are provided above.

Encompassed by the present disclosure are pharmaceutical kits containingthe anti-PG antibodies (including antibody conjugates) of thedisclosure. The pharmaceutical kit is a package comprising the anti-PGantibody composition (e.g., either in lyophilized form or as an aqueoussolution) and one or more of the following:

-   -   A second agent, for example as described above;    -   A device for administering an anti-PG antibody composition, for        example a pen, needle and/or syringe; and    -   Pharmaceutical grade water or buffer to re-suspend the antibody        if the antibody is in lyophilized form.

Each unit dose of anti-PG antibody can be packaged separately, and a kitcan contain one or more unit doses (e.g., two unit doses, three unitdoses, four unit doses, five unit doses, eight unit doses, ten unitdoses, or more). In a specific embodiment, the one or more unit dosesare each housed in a syringe or pen.

7.4 Effective Dosages And Treatment Regimens

The anti-PG antibodies of the present disclosure are administered to thesubject in an amount sufficient or effective to provide a therapeuticbenefit. In the context of preventing gastrointestinal cancer, includingCRC, in a subject predisposed to develop adenomatous polyps, atherapeutic benefit can be inferred if one or more of the following isachieved: reduction or lack of increase in the number and/or size ofpolyps in a subject; absence of malignant tumors, including where asubject has or had polyps; reduction or lack of increase in plasma orserum PG level; regression from a more advanced stage of polyposis to aless advanced stage of polyposis, according to Spigelman'sclassification (e.g., regression from Stage IV to Stage III, from StageIII to Stage II, from Stage II to Stage I); lack of progression fromSpigelman Stage IV polyposis to gastrointestinal cancer. Pharmaceuticalcompositions comprising anti-PG antibodies can be administered toindividuals (e.g., human subjects) at effective dosages.

Complete prevention of gastronintestinal cancer, while desirable, is notrequired for therapeutic benefit to exist. Indeed, as most patientssuffering from FAP require major surgery by the age of 25, slowing theprogression of the disease such that surgery can be delayed improvesquality of life. Furthermore, any delay in the onset of gastrointestinalcancer, such as CRC, provides a therapeutic benefit.

In some contexts, therapeutic benefit can be correlated with one or moresurrogate end points, in accordance with the knowledge of one ofordinary skill in the art. By way of example and not limitation, plasmaand/or serum PG concentrations can be measured in a subject over time,with a reduction in PG levels, or a level below a threshold level, forexample, below about 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, or 5 pM, beingindicative of therapeutic benefit.

Polyp size and number can be measured using endoscopic techniques, suchas colonoscopy, as well as other methods known to those of ordinaryskill in the art.

Binding all free PG is not required to achieve therapeutic efficacy,although it may be desirable. Free PG means PG that is available to bebound by an anti-PG antibody. Rather, reducing the concentration of freePG within or around polyps, systemically, in particular body fluids, orelsewhere, to a more limited extent may also be effective. Exemplarytissues and body fluids in which free PG concentration may be reduced byadministration of anti-PG antibody(ies) compositions include, but arenot limited to, polyp or tumor samples removed from a patient, ascitesfluid, fluid from pleural effusions, cerebrospinal fluid, lymph, blood,plasma, serum and others. The concentration of PG in one or more ofthese tissues or body fluids can be quantified using an ELISA techniqueor other techniques familiar to those of ordinary skill in the art.

In accordance with the knowledge of those ordinarily skilled in the art,the dose of an anti-PG antibody can be titrated in a patient so as toreduce the free PG concentration in a tissue or body fluid of interestat a predetermined time after administration at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90, or 100%, or about 5%-10%, about10%-15%, about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%,about 35%-40%, about 40%-45%, about 45%-50%, about 50%-55%, about55%-60%, about 60%-65%, about 65%-70%, about 70%-75%, about 75%-80%,about 80%-85%, about 85%-90%, or about 90%-95%, or a percentagereduction in free PG concentration ranging between any of the foregoingvalues.

The amount of anti-PG antibody administered will depend on a variety offactors, including the number and size of adenomatous polyps found inthe subject, the form, route and site of administration, the treatmentregimen (e.g., whether a second therapeutic agent is used), the age andcondition of the particular subject being treated, the sensitivity ofthe patient to anti-PG antibodies. The appropriate dosage can be readilydetermined by a person skilled in the art. Ultimately, a physician willdetermine appropriate dosages to be used. This dosage can be repeated asoften as appropriate. If side effects develop the amount and/orfrequency of the dosage can be altered or reduced, in accordance withnormal clinical practice. The proper dosage and treatment regimen can beestablished by monitoring the progress of treatment using conventionaltechniques known to the people skilled of the art.

Effective dosages can be estimated initially from in vitro assays. Forexample, an initial dose for use in animals may be formulated to achievea circulating blood or serum concentration of anti-PG antibody that isat or above the binding affinity of the antibody for progastrin asmeasured in vitro. Calculating dosages to achieve such circulating bloodor serum concentrations taking into account the bioavailability of theparticular antibody is well within the capabilities of skilled artisans.For guidance, the reader is referred to Fingl & Woodbury, “GeneralPrinciples” in Goodman and Gilman's The Pharmaceutical Basis ofTherapeutics, Chapter 1, latest edition, Pagamonon Press, and thereferences cited therein.

Initial dosages can be estimated from in vivo data, such as animalmodels. Animal models useful for testing the efficacy of compounds todelay or prevent development of gastrointestinal tumors, including CRCtumors, are well known in the art. Additionally, an animal model of FAPis described in the Examples below. Ordinarily skilled artisans canroutinely adapt such information to determine dosages suitable for humanadministration.

In specific embodiments, an i.v. dose may be determined for anindividual subject by measuring the serum or plasma PG concentration ofthe individual a few times a few days to a few weeks prior to treatmentand calculating an amount of anti-PG antibody that would be saturating,i.e., an amount that would be sufficient to bind all of the PG. As willbe appreciated by skilled artisans, the amount of any specific antibodynecessary to achieve saturation for a given serum or plasmaconcentration of PG will depend, in part, on the affinity constant ofthe particular antibody. Methods for calculating saturating quantitiesfor specific anti-PG antibodies of interest are well-known.

To insure saturation, an amount that is greater than the calculatedsaturating amount may be administered, for example, at least 2-, 3-, 4-,5-, 6-, 7-, 8-, 9- or even 10-fold greater than the calculatedsaturating amount may be administered. For modes of administration otherthan i.v., the amount can be adjusted based upon pharmacokinetic andbioavailability, as is well known in the art.

The effective dose of an anti-PG antibody of the disclosure can rangefrom about 0.001 to about 75 mg/kg per single (e.g. bolus)administration, multiple administrations or continuous (e.g. infusion)administration, or to achieve a serum concentration of 0.01-5000 μg/mlserum concentration per single administration, multiple administrationsor continuous administration, or any effective range or value thereindepending on the condition being treated, the route of administrationand the age, weight and condition of the subject. In certainembodiments, each dose can range from about 0.1 mg/kg to about 0.5mg/kg; about 0.25 mg/kg to about 0.75 mg/kg; about 0.5 mg/kg to about 1mg/kg; about 1 mg/kg to about 2 mg/kg; about 1.5 mg/kg to about 2.5mg/kg; about 2 mg/kg to about 3 mg/kg; about 2.5 mg/kg to about 3.5mg/kg; about 3 mg/kg to about 4 mg/kg; about 3.5 mg/kg to about 4.5mg/kg; about 4 mg/kg to about 5 mg/kg; about 5 mg/kg to about 7 mg/kg;about 6 mg/kg to about 8 mg/kg; about 7 mg/kg to about 9 mg/kg; about 8mg/kg to about 10 mg/kg; about 10 mg/kg to about 15 mg/kg; about 12.5mg/kg to about 17.5 mg/kg; about 15 mg/kg to about 20 mg/kg; about 17.5mg/kg to about 22.5 mg/kg; about 20 mg/kg to about 25 mg/kg; about 22.5mg/kg to about 27.5 mg/kg; about 25 mg/kg to about 30 mg/kg; about 30mg/kg to about 40 mg/kg; about 35 mg/kg to about 45 mg/kg; about 40mg/kg to about 50 mg/kg; about 45 mg/kg to about 55 mg/kg; about 50mg/kg to about 60 mg/kg; about 55 mg/kg to about 65 mg/kg; about 60mg/kg to about 70 mg/kg; about 65 mg/kg to about 75 mg/kg. Other dosageranges are also possible.

Amount, frequency, and duration of administration will depend on avariety of factors, such as the patient's age, weight, and diseasecondition. Anti-PG treatment is indicated in subjects in whompre-cancerous adenomatous polyps are detected and/or removed, andsubjects diagnosed with FAP who have yet to manifest polyps. In humanswith FAP, polyps generally begin to appear in the second decade. Anti-PGtreatment can be initiated before or at the time polyps are detected insubjects with FAP. For subjects with sporadic adenomatous polyposis,anti-PG treatment can be initiated at the time polyps are detected.Anti-PG treatment can also be initiated in subjects who have had atleast one polyp removed, and therefore are at increased risk ofdeveloping more polyps and gastrointestinal cancer, including CRC.

A treatment regimen for administration can continue for 2 weeks toindefinitely. Optionally, the treatment regimen provides for repeatedadministration, e.g., once daily, twice daily, every two days, threedays, five days, one week, two weeks, or one month. The repeatedadministration can be at the same dose or at a different dose. Theadministration can be repeated once, twice, three times, four times,five times, six times, seven times, eight times, nine times, ten times,or more. An effective amount of anti-PG antibody can be administered asa single dose or over the course of a treatment regimen. The duration ofanti-PG treatment for patients predisposed to develop adenomatouspolyposis is preferably long, e.g., over the course of years, but may beshorter, e.g., one to several months, to a year.

7.5 Methods of Selecting Patients for Follow-Up or Treatment, andPatient Monitoring to Determine Treatment Efficacy

Without wishing to be bound by any particular theory of operation, it isbelieved that elevated levels of PG are associated with thetransformation of adenomatous polyps from benign to malignant. As shownin Example 6 below, subjects with multiple polyps had elevated PG levelswhereas subjects who did not have polyps had either low or undetectablelevels of serum PG. Based on this observation, plasma and/or serumlevels of PG can be used to identify patients for follow-up ortreatment, as well as to monitor the effectiveness of prophylaxis inpatients undergoing treatment.

Monitoring PG levels in individuals with FAP or a history of sporadicadenomatous polyposis is useful for identifying subjects in whom followup by colonoscopy is warranted, as well as patients in need of anti-PGtreatment. Standard care for individuals predisposed to developingadenomatous polyps is endoscopy at an interval of 3 to 5 years. Thisinterval between testing may mean that, in certain individuals, numerouspolyps have developed or cancer has set in by the time follow upendoscopy is performed. A simple blood test for PG levels is readilyperformed at more frequent intervals and can identify those individualswho should undergo endoscopy (e.g., colonoscopy) sooner or who arecandidates for anti-PG treatment.

An individual diagnosed with FAP or in whom polyps have previously beendetected can be monitored to determine PG level, or concentration, in abodily fluid, such as whole blood, plasma, or serum, relative to anappropriate baseline. Accordingly, a PG level is measured in a samplefrom the individual and then compared to a baseline PG level.

Where the PG level in a subject with FAP or a history of sporadicadenomatous polyposis is unchanged relative to previous measurements inthe subject or equal to a baseline level for the relevant population towhich the subject is being compared, the subject is scored as notrequiring further follow up. By contrast, where the PG concentration isabove the baseline, or is seen to rise over a period of time in thesubject, the subject is a candidate for further follow-up, including,for example, colonoscopy, and for anti-PG treatment.

For purposes of monitoring efficacy of the treatment, blood, plasma, orserum PG levels can be measured in the patient receiving anti-PGtreatment at specified time points, and used as an indication of whetherthe treatment is effective based on whether the measured level is aboveor below a baseline PG level. This information can be used by careproviders to decide whether to continue administering an anti-PGantibody or modify treatment. These methods can be used to monitoranti-PG treatment, used alone, or in combination with other treatments,as described above.

In some embodiments of the methods, the PG level in one or more bodilyfluids, such as whole blood, plasma, serum, of a patient receivinganti-PG antibody treatment can be measured and then compared to abaseline level. A decrease in concentration over time, and/or a measuredlevel below a threshold value at a particular point in time, isindicative of efficacy. An increase in concentration over time and/orabove-baseline PG level is indicative of lack of treatment efficacy.Typically, PG level is the concentration of PG in the sample, expressedin molar (M) amounts or moles/liter (mol/liter).

The baseline level can be a single number or a range of numbers. Thebaseline can be based on one or more measurements taken from the patientor based on measurements of PG in samples from a population ofindividuals. In some embodiments of the methods, the baseline is a PGlevel from the same patient, taken at one or more interval, for example,before the initiation of anti-PG treatment, during the course oftreatment, or after treatment has been stopped. In some embodiments, thebaseline can be an average PG level in a population of individuals withcharacteristics similar to those of the individual undergoingmonitoring. Such characteristics may include, but are not necessarilylimited to sex, age, location of mutation in APC gene, stage inSpigelman classification, history of surgery, anti-PG treatment, orother treatment. In some embodiments, the baseline is a specific PGlevel, such as about 50 pM, about 40 pM, about 30 pM, about 20 pM, about10 pM, about 5 pM, about 2 pM, about 1 pM, or even lower. In someembodiments, the baseline is a range.

PG levels can be measured using techniques familiar to those of ordinaryskill in the art, such as, but not limited to, RIA and ELISA. In aspecific embodiment, PG levels may be measured using a sandwich ELISAwith one anti-PG antibody targeting the N-terminus of progastrin and asecond anti-PG antibody targeting the C-terminus of progastrin.Exemplary N- and C-terminal anti-PG antibodies useful for such asandwich assay are described in a later section. In such an assay, asurface, such as the wells in a 96-well plate, is prepared to which aknown quantity of a first, “capture,” N-terminal or C-terminal anti-PGantibody is bound. A test sample is then applied to the surface followedby an incubation period. The surface is then washed and a solutioncontaining a second, “detection,” anti-PG antibody is applied, where thedetection antibody binds a different epitope of PG (for example, if thecapture antibody is a C-terminal anti-PG antibody, an N-terminal anti-PGantibody is used as the detection antibody, and vice versa). PG levelsare then measured either directly (if, for example, the detectionantibody is conjugated to a detectable label) or indirectly (through alabeled secondary antibody that binds the detection anti-PG antibody).For this assay, antibodies should be used in excess such that all PG isbound and quantified. A specific sandwich assay for measuring plasmaand/or serum PG levels is provided in Example 1.

Multiple measurements at different intervals may be taken, and thengraphed to determine if a trend exists. In a non-limiting example, PGlevels can be determined at weekly, monthly, or annual intervals while apatient is received anti-PG antibodies. Other intervals are alsopossible.

In an embodiment involving a round of therapy using an anti-PG antibody,one or more measurements may also be taken during the course of therapyso that the effect of the antibodies on PG levels can be estimated. Inother such embodiments, where residual anti-PG antibodies are present ina patient during sampling, the data may show a reduction in PG levels,due to sequestration of PG by the antibodies, followed by a rise, asthis effect abates, followed by a subsequent decline, if the treatmentwas effective. In yet other embodiments, post-therapy measurements canbe taken after it is estimated that the anti-PG antibodies have beencleared from the patient so that binding of PG by such antibodies doesnot affect the accuracy of the measurement of PG concentration.

Because eating usually increases gastrin synthesis and secretion, it mayalso cause transient increases in blood PG levels, which may interferewith the accurate measurement of PG levels in patients being monitored.To avoid this effect, particularly where PG concentration in bloodsamples is to be determined, samples can be taken from the patient afterfasting.

7.6 Anti-PG Antibodies

Antibodies useful in the methods disclosed herein are those thatspecifically bind progastrin over other products of the gastrin gene.Referring to FIG. 1, the human gastrin gene is translated into a101-amino acid polypeptide, called pre-progastrin, which contains asignal sequence (underlined) that is cleaved, giving rise to humanprogastrin, an 80-amino-acid polypeptide. Progastrin, in turn, iscleaved to generate a 34-amino-acid product, corresponding in sequenceto residues 38-71 of progastrin, which is then extended at its carboxyterminus with a glycine residue, generating glycine-extended G34(“G34-Gly”). A by-product of this cleavage is a 6-amino-acid peptide,called the C-terminal flanking peptide, or CTFP, which corresponds insequence to residues 75-80 of progastrin. G34-Gly is then furthercleaved to generate a 17-residue polypeptide corresponding in sequenceto residues 55-71 of progastrin and referred to as G17-Gly. Removal ofthe C-terminal glycines of G34-Gly and G17-Gly, followed by C-terminalamidation, yields G34 and G17, respectively, both of which areC-terminal amidated.

As used herein, an antibody is “highly specific for” hPG or “highlyspecifically binds” hPG if it binds to full-length progastrin but doesnot bind at all to CTFP, to amidated gastrin, or to glycine-extendedgastrin, and is “specific for” hPG or “specifically binds” hPG if itexhibits at least about 5-fold greater binding of hPG than CTFP and theother products of the gastrin gene, as measured in standard bindingassays. A specific ELISA assay that can be used to assess thespecificity of a particular anti-hPG antibody is provided in Example 2.

Such highly specific and/or specific anti-hPG antibodies (referred toherein as “anti-hPG antibodies”) may be polyclonal (“anti-hPG PAbs”) ormonoclonal (“anti-hPG MAbs”), although for therapeutic uses and, in someinstances, diagnostic or other in vitro uses, monoclonal antibodies arepreferred.

The epitope bound by the anti-hPG antibodies is not critical. Usefulanti-hPG antibodies may bind an N-terminal region of hPG, a C-terminalregion of hPG, or a different region of hPG. Recently, it has beendiscovered that, at least for monoclonal anti-hPG antibodies, theselection of antigen used to raise the anti-hPG antibodies may beimportant (see, International Application No. PCT/EP2010/006329 filedOct. 15, 2010 and U.S. application Ser. No. 12/906,041 filed Oct. 15,2010, the disclosures and specifically disclosed anti-hPG antibodies ofwhich are incorporated herein by reference; hereinafter referred to asthe '329 and '041 applications, respectively). As disclosed in the '329and '041 applications, not all antigens derived from hPG stimulateproduction of monoclonal antibodies that specifically bind hPG underphysiological conditions. Indeed, certain antigens that have been usedto successfully raise polyclonal anti-hPG antibodies, such asfull-length recombinant hPG (see, e.g., WO 08/076,454 to Singh) and apeptide corresponding to the last ten amino acids at the C-terminal endof hPG (see WO 07/135,542 to Hollande et al.) failed to generatemonoclonal antibodies. As noted in the '329 and '041 applications,antigenic N-terminal and C-terminal sequences within the hPG sequencehave been identified that can be used to generate nonoclonal antibodiesthat specifically bind hPG. Interestingly, the antigenic sequence neednot be limited to regions of the hPG sequence that are unique to it.Peptide antigens having regions of sequence in common with otherproducts of the gastrin gene, for example, G17, G34 and CTFP, yieldmonoclonal antibodies that not only bind hPG, but bind it specifically.

Anti-hPG antibodies obtainable using a peptide antigen having a sequencecorresponding to an N-terminal region of hPG and/or that bind anN-terminal region of hPG are referred to herein as “N-terminal anti-PGantibodies.” A specific exemplary antigenic region of hPG that can beused to construct an immunogen suitable for obtaining both polyclonaland monoclonal antibodies specific for hPG corresponds to residue 1 to14 of hPG: SWKPRSQQPDAPLG (SEQ ID NO:25). Exemplary immonogens usefulfor obtaining N-terminal anti-hPG antibodies, as well as CDR and V_(H)and V_(L) sequences of N-terminal anti-hPG monoclonal antibodiesobtained with these exemplary immunogens, are provided in TABLE 2A,below, and the Example sections:

TABLE 2A N-Terminal Anti-hPG Monoclonal Antibodies Hybridoma Immunogen(Deposit #) MAb Murine CDR Sequences N1 43B9G11 MAb1 N1 WE5H2G7 MAb2 N26B5B11C10 MAb3 V_(H) CDR 1.3 GYIFTSYW (SEQ ID NO: 1) V_(H) CDR 2.3FYPGNSDS (SEQ ID NO: 2) V_(H) CDR 3.3 TRRDSPQY (SEQ ID NO: 3) V_(L) CDR1.3 QSIVHSNGNTY (SEQ ID NO: 4) V_(L) CDR 2.3 KVS (SEQ ID NO: 5) V_(L)CDR 3.3 FQGSHVPFT (SEQ ID NO: 6) N2 20D2C3G2 MAb4 V_(H) CDR 1.4 GYTFSSSW(SEQ ID NO: 7) V_(H) CDR 2.4 FLPGSGST (SEQ ID NO: 8) V_(H) CDR 3.4ATDGNYDWFAY (SEQ ID NO: 9) V_(L) CDR 1.4 QSLVHSSGVTY (SEQ ID NO: 10)V_(L) CDR 2.4 KVS (SEQ ID NO: 5) V_(L) CDR 3.4 SQSTHVPPT (SEQ ID NO: 11)N2 1E9A4A4 MAb15 (1-4376) N2 1E9D9B6 MAb16 V_(H) CDR 1.16 GYTFTSYY (SEQID NO: 39) V_(H) CDR 2.16 INPSNGGT (SEQ ID NO: 43) V_(H) CDR 3.16TRGGYYPFDY (SEQ ID NO: 47) V_(L) CDR 1.16 QSLLDSDGKTY (SEQ ID NO: 50)V_(L) CDR 2.16 LVS (SEQ ID NO: 53) V_(L) CDR 3.16 WQGTHSPYT (SEQ ID NO:57) N2 1C8D10F5 MAb17 N2 1A7C3F11 MAb18 N2 1B3B4F11 MAb19 V_(H) CDR1.19GYSITSDYA (SEQ ID NO: 40) V_(H) CDR2.19 ISFSGYT (SEQ ID NO: 44) V_(H)CDR3.19 AREVNYGDSYHFDY (SEQ ID NO: 48) V_(L) CDR1.19 SQHRTYT (SEQ ID NO:51) V_(L) CDR2.19 VKKDGSH (SEQ ID NO: 54) V_(L) CDR3.19 GVGDAIKGQSVFV(SEQ ID NO: 58) N2 1C11F5E8 MAb20 Hybridoma Humanized V_(H) and V_(L)Immunogen (Deposit #) MAb Murine V_(H) and V_(L) Sequences Sequences(projected) N1 43B9G11 MAb1 N1 WE5H2G7 MAb2 N2 6B5B11C10 MAb3 mV_(H).3(SEQ ID NO. 12) hV_(H).3 (SEQ ID NO: 21) mV_(L).3 (SEQ ID NO: 13)hV_(L).3 (SEQ ID NO: 22) N2 20D2C3G2 MAb4 mV_(H).4 (SEQ ID NO: 14)hV_(H).4 (SEQ ID NO: 23) mV_(L).4 (SEQ ID NO: 15) hV_(L).4 (SEQ ID NO:24) N2 1E9A4A4 MAb15 (1-4376) N2 1E9D9B6 MAb16 mV_(H).16 (SEQ ID NO: 61)hV_(H).16a (SEQ ID NO: 84) hV_(H).16b (SEQ ID NO: 86) hV_(H).16c (SEQ IDNO: 88) mV_(L).16 (SEQ ID NO: 65) hV_(L).16a (SEQ ID NO: 85) hV_(L).16b(SEQ ID NO: 87) hV_(L).16c (SEQ ID NO: 89) N2 1C8D10F5 MAb17 N2 1A7C3F11MAb18 N2 1B3B4F11 MAb19 mV_(H).19 (SEQ ID NO: 62) hV_(H).19a (SEQ ID NO:90) hV_(H).19b (SEQ ID NO: 92) hV_(H).19c (SEQ ID NO: 94) mV_(L).19 (SEQID NO: 66) hV_(L).19a (SEQ ID NO: 91) hV_(L).19b (SEQ ID NO: 93)hV_(L).19c (SEQ ID NO: 95) N2 1C11F5E8 MAb20 Immunogen N1 =SWKPRSQQPDAPLG-Ahx-Cys-BSA, also represented as (SEQ ID NO:25)-Ahx-Cys-BSA Immunogen N2 = SWKPRSQQPDAPLG-Ahx-Cys-KLH, alsorepresented as (SEQ ID NO: 25)-Ahx-Cys-KLH In TABLE 2A, all amino acidsequences are represented using conventional N→C orientation. For eachimmunogen, the progastrin peptide was synthesized with a C-terminallinker of one aminohexanoic acid (Ahx) residue followed by a cysteine(Cys) residue, which was then conjugated to a either a bovine serumalbumin (“BSA”) or keyhole limpet hemocyanin (“KLH”) carrier via the Cyslinker residue.

Anti-hPG antibodies obtainable using a peptide antigen having a sequencecorresponding to a C-terminal region of hPG, and/or that bind aC-terminal region of hPG, are referred to herein as “C-terminal anti-hPGantibodies.” A specific exemplary antigenic region that can be used toconstruct an immunogen useful for obtaining both polyclonal andmonoclonal C-terminal anti-hPG antibodies corresponds to residues 55 to80 of hPG: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO:27). Exemplaryimmunogens including this antigen useful for obtaining C-terminalanti-hPG antibodies, as well as CDR and V_(H) and V_(L) sequences ofC-terminal anti-hPG monoclonal antibodies obtained with these exemplaryimmunogens, are provided in TABLE 2B, below, and the Examples section.

TABLE 2B C-Terminal Anti-hPG Monoclonal Antibodies Immu- HybridomaMurine V_(H) Humanized V_(H) and V_(L) nogen (Deposit #) MAb Murine CDRSequences and V_(L) Sequences Sequences (projected) C1 1B4A11D11 MAb5(1-4371) C1 1B6A11F2 MAb6 (1-4372) C1 1B11E4B11 MAb7 (1-4373) C11C10D3B9 MAb8 V_(H) CDR 1.8 GFTFTTYA (SEQ ID NO: 37) mV_(H).8 (SEQ IDNO: 59) hV_(H).8a (SEQ ID NO: 75) V_(H) CDR 2.8 ISSGGTYT (SEQ ID NO: 41)hV_(H).8b (SEQ ID NO: 77) V_(H) CDR 3.8 ATQGNYSLDF (SEQ ID NO: 45)hV_(H).8c (SEQ ID NO: 79) V_(L) CDR 1.8 KSLRHTKGITF (SEQ ID NO: 49)mV_(L).8 (SEQ ID NO: 63) hV_(L).8a (SEQ ID NO: 76) V_(L) CDR 2.8 QMS(SEQ ID NO: 52) hV_(L).8b (SEQ ID NO: 78) V_(L) CDR 3.8 AQNLELPLT (SEQID NO: 55) hV_(L).8c (SEQ ID NO: 76) C1 1D8F5B3 MAb9 C1 1E1C7B4 MAb10 C12B4C8C8 MAb11 (1-4374) C1 2B11E6G4 MAb12 (1-4375) C1 2C6C3C7 MAb13 V_(H)CDR 1.13 GFIFSSYG (SEQ ID NO: 38) mV_(H).13 (SEQ ID NO: 60) hV_(H).13a(SEQ ID NO: 80) V_(H) CDR 2.13 INTFGDRT (SEQ ID NO: 42) hV_(H).13b (SEQID NO: 82) V_(H) CDR 3.13 ARGTGTY (SEQ ID NO: 46) V_(L) CDR 1.13QSLLDSDGKTY (SEQ ID NO: 50) mV_(L).13 (SEQ ID NO: 64) hV_(L).13a (SEQ IDNO: 81) V_(L) CDR 2.13 LVS (SEQ ID NO: 53) hV_(L).13b (SEQ ID NO: 83)V_(L) CDR 3.13 WQGTHFPQT (SEQ ID NO: 56) C1 2H9F4B7 MAb14 C2 1F11F5E10MAb21 C2 1F11F5G9 MAb22 C2 1A11F2C9 MAb23 Immunogen C1 =KLH-Cys-Ahx-Ahx-QGPWLEEEEEAYGWMDFGRRSAEDEN, also represented asKLH-Cys-Ahx-Ahx-(SEQ ID NO: 27) Immunogen C2 =DT-Cys-Ahx-Ahx-QGPWLEEEEEAYGWMDFGRRSAEDEN, also represented asDT-Cys-Ahx-Ahx-(SEQ ID NO: 27) In TABLE 2B, all amino acid sequences arerepresented using conventional N→C orientation. For each immunogen, theprogastrin peptide was synthesized with an N-terminal Ahx-Ahx-Cyslinker, which was then conjugated to a either a keyhole limpethemocyanin (“KLH”) or a diphtheria toxin (“DT”) carrier via the Cyslinker residue.

The specific epitopes bound by the exemplary anti-hPG monoclonalantibodies MAb1-MAb23 provided in TABLES 2A and 2B were mapped using theSPOT technique and alanine scanning, as described in Laune et al., 2002,J. Immunol. Methods 267:53-70 and Laune, 1997, J. Biol. Chem.272:30937-30944, respectively (see also, Example 6 of the '329application).

In the SPOT technique, 15 amino acid peptide sequences spanning aputative epitope are generated and spotted onto a nitrocellulosemembrane which is then probed with the test antibody to determine theminimal epitope sequence recognized by the antibody. Alanine scanning isused to determine residues within an epitope that are critical forantibody binding. Each residue within a putative epitope is mutated, oneby one, to an alanine, and the alanine-containing peptides are thenprobed with the test antibody.

For N-terminal anti-hPG monoclonal antibodies MAbs1-4 and 15-20,epitopes comprise at least the following sequences: DAPLG (SEQ IDNO:28), PDAPLG (SEQ ID NO:29), PRSQQPD (SEQ ID NO:30), WKPRSQQPD (SEQ IDNO:31), or WKPRSQQPDAPLG (SEQ ID NO:32), as shown in TABLE 3A below.

TABLE 3A MAb PG peptide antigen: SEQ ID # SWKPRSQQPDAPLG NO MAb2WKPRSQQPDAPLG 32 MAb4 WKPRSQQPDAPLG 32 MAb1 PDAPLG 29 MAb3 DAPLG 28MAb17 WKPRSQQPD 31 MAb18 WKPRSQQPD 31 MAb19 WKPRSQQPD 31 MAb20 WKPRSQQPD31 MAb15 PRSQQPD 30 MAb16 PRSQQPD 30

For C-terminal anti-hPG monoclonal antibodies MAbs5-7, 9-12, 14 and21-23, epitopes comprise at least the following sequences: FGRR (SEQ IDNO:33), MDFGR (SEQ ID NO:34), AEDEN (SEQ ID NO:35), and GWMDFGRR (SEQ IDNO:36), as shown in TABLE 3B, below.

TABLE 3B MAb PG peptide antigen: SEQ ID # QGPWLEEEEEAYGWMDFGRRSAEDEN NOMAb14 GWMDFGRR 36 MAb11 MDFGR 34 MAb5 FGRR 33 MAb6 FGRR 33 MAb7 FGRR 33MAb9 FGRR 33 MAb10 FGRR..E 33 MAb12 FGRR 33 MAb23 AEDEN 35

The epitope mapping experiments reveal that anti-hPG MAb2 and MAb4 bindthe same epitope; anti-hPG MAb1 and MAb3 bind approximately the sameepitope; MAb17, MAb18, MAb19, and MAb20 bind approximately the sameepitope; MAb15 and MAb16 bind approximately the same epitope; anti-hPGMAb5, MAb6, MAb7, MAb9, and MAb12 bind the same epitope and bindapproximately the same epitope as anti-hPG MAb10; and anti-hPG MAb11 andMAb14 bind approximately the same epitope.

Specific embodiments of N-terminal anti-PG antibodies useful in themethods and kits described herein include antibodies that bind anepitope that includes residues 10 to 14 of hPG (SEQ ID NO:28), residues9 to 14 of hPG (SEQ ID NO:29), residues 4 to 10 of hPG (SEQ ID NO:30),residues 2 to 10 of hPG (SEQ ID NO:31), or residues 2 to 14 of hPG (SEQID NO:32).

Specific embodiments of C-terminal anti-PG antibodies useful in themethods and kits described herein include antibodies that bind anepitope that includes residues 71 to 74 of hPG (SEQ ID NO:33), residues69 to 73 of hPG (SEQ ID NO:34), residues 76 to 80 of hPG (SEQ ID NO:35),or residues 67 to 74 of hPG (SEQ ID NO:36).

N-terminal and C-terminal anti-hPG antibodies useful in the methods andkits disclosed herein in addition to those provided in TABLES 2A & 2Bcan be identified in competitive binding assays with exemplary MAbs1-23, or with other reference antibodies that bind N- or C-terminalepitopes, as will be described in more detail in a later section.

As also reported in the '329 and '041 applications, not all anti-hPGantibodies, even those that exhibit a high degree of specificity andaffinity for hPG, may neutralize the biological activity of hPG. Forexample, although anti-hPG MAb14 binds hPG with a K_(D) of about 6 pM,it did not inhibit the growth of colorectal cancer cells in an in vitroassay, whereas other anti-hPG monoclonal antibodies exhibitedsignificant inhibitory activity (see, e.g., Example 7 of the '329application). While both non-neutralizing and neutralizing antibodiesthat specifically bind hPG are useful for the various diagnostic andmonitoring methods described herein, anti-hPG antibodies useful fortherapeutic methods should exhibit neutralizing activity.

As used herein, a “neutralizing anti-hPG antibody” is an anti-hPGantibody that yields a statistically significant reduction in the numberof live LS 174T in a test sample treated with the anti-hPG antibody ascompared to a control sample treated with a non-specific antibody. Aspecific assay for assessing the ability of any particular anti-hPGantibody to neutralize hPG is described in Example 3. Those anti-hPGantibodies that exhibit at least about a 50% reduction in the number oflive cells in this assay are believed to be especially useful in methodsof preventing gastrointestinal cancer, including CRC, although anti-hPGantibodies exhibiting lower levels of neutralizing activity, forexample, a statistically significant reduction of 40%, 30%, 20%, 15%, oreven 10%, in the number of live cells in this assay, are expected toprovide therapeutic benefits.

Accordingly, in some embodiments, for example therapeutic embodiments,useful anti-hPG antibodies are neutralizing. As disclosed in the '329and '041 applications, the ability of an anti-hPG monoclonal antibody isnot epitope-dependent, as both N-terminal and C-terminal anti-hPGmonoclonal antibodies exhibited neutralizing activity in assays withcolorectal cancer cellsbearing a mutation in the APC gene. Thus, in somespecific embodiments, the neutralizing anti-hPG antibodies areN-terminal neutralizing anti-hPG antibodies. In other embodiments, theneutralizing anti-hPG antibodies are C-terminal neutralizing anti-hPGantibodies.

The affinity of any specific anti-hPG antibody is not critical. However,for some uses, antibodies exhibiting affinities of at least about 1 μMmay be preferred. For therapeutic uses, an affinity of at least about 90nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 7 nM,6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even greater,may be desirable. The measured affinities of the anti-hPG monoclonalantibodies identified in TABLES 2A & 2B range from 10⁻⁶ to 10⁻¹² M, asnoted in TABLE 4, below:

TABLE 4 MAb# Affinity (measured K_(D)) MAb1 2.5 μM (2.5 × 10⁻⁶ M) MAb2185 nM (1.85 × 10⁻⁷ M) MAb3 6.4 nM (6.4 × 10⁻⁹ M) MAb4 3.5 nM (3.5 ×10⁻⁹ M) MAb5 13 pM (1.30 × 10⁻¹¹ M) MAb6 0.6 nM (6.38 × 10⁻¹⁰ M) MAb7 58pM (5.84 × 10⁻¹¹ M) MAb8 0.1 nM (1.08 × 10⁻¹⁰ M) MAb10 3.6 nM (3.62 ×10⁻⁹ M) MAb11 0.3 nM (3.12 × 10⁻¹⁰ M) MAb12 0.4 nM (4.43 × 10⁻¹⁰ M)MAb13 0.6 nM (6.12 × 10⁻¹⁰ M) MAb14 6.8 pM (6.86 × 10⁻¹² M) MAb15 0.2 nM(2.11 × 10⁻¹⁰ M) MAb16 0.2 nM (2.78 × 10⁻¹⁰ M) MAb17 8.3 nM (8.29 × 10⁻⁹M) MAb18 1.2 nM (1.24 × 10⁻⁹ M) MAb19 0.7 nM (7.79 × 10⁻¹⁰ M) MAb20 0.2nM (2.47 × 10⁻¹⁰ M) MAb21 3.9 nM (3.90 × 10⁻⁹ M) MAb22 5 nM (4.94 × 10⁻⁹M) MAb23 0.4 μM (3.99 × 10⁻⁷ M)

An anti-PG monoclonal antibody having an affinity especially suited fora particular desired application can be readily selected from amongstthese, or generated or designed using the various immunogens,complementarity determining region (CDR) sequences, variable heavy(V_(H)) and variable light (V_(L)) chain sequences of anti-hPGantibodies described herein. The affinity of any particular anti-PGmonoclonal antibody can be determined using techniques well known in theart or described herein, such as for example, ELISA, isothermaltitration calorimetry (ITC), BIAcore, or fluorescent polarizationassays. A specific assay is provided in Example 4.

As noted in TABLES 2A & 2B, several N-terminal and C-terminal monoclonalanti-hPG antibodies have been identified. All of these antibodies arespecific for hPG, and, with the exception of MAb14, all exhibitedneutralizing activity in tests with colorectal cancer cells. Several ofthe hybridomas useful for obtaining the antibodies were deposited onOct. 6, 2010 with the Collection Nationale de Cultures de Microorganisms(CNCM) in accordance with the Treaty of Budapest. The designated namesof the hybridomas producing anti-hPG MAbs1-23 and the depositoryregistration numbers of those hybridomas deposited are provided inTABLES 2A & 2B. In addition, for several of the antibodies, the aminoacid sequences of their variable heavy chains (V_(H)), variable lightchains (V_(L)), V_(L) complementarity determining regions (CDRs) andV_(H) CDRs have been determined. These amino acid sequences, and theshorthand nomenclature used to reference them throughout the disclosure,are also provided in TABLES 2A & 2B. Briefly, murine heavy and lightchain variable domains are referred to herein as mV_(H) and mV_(L)followed by the number of the corresponding monoclonal antibody, forexample mV_(H).3 and mV_(L).3 for the variable light and variable heavychains of anti-hPG MAb3, respectively. Similarly, human heavy and lightchain variable domains are referred to herein as hV_(H) and hV_(L)followed by the number of the corresponding monoclonal antibody. Thethree variable heavy chain CDRs and three variable light chain CDRs arereferred to as V_(H) CDR 1, 2, or 3, and V_(L) CDR 1, 2, or 3,respectively, followed by the number of the specific anti-hPG monoclonalantibody. For example, V_(H) CDR 1 of MAb3 is denoted V_(H) CDR 1.3 andV_(L) CDR 1 of MAb3 is denoted V_(L) CDR 1.3. V_(H) CDR 2 of MAb3 isdenoted V_(H) CDR 2.3, and V_(L) CDR 2 of MAb3 is denoted V_(L) CDR 2.3.

It is expected that corresponding CDRs and/or V_(H) and V_(L) chains ofanti-hPG monoclonal antibodies that bind approximately the same epitopescould be interchanged to yield new anti-hPG monoclonal antibodies usefulin the methods and kits described herein. For example, as noted above,exemplary anti-hPG monoclonal antibodies MAb5 and MAb6 bind the sameepitope. An anti-hPG monoclonal antibody can be designed that includes,in its V_(L) chain, various combinations of the V_(L) CDRs of these twoantibodies, and/or in its V_(H) chain various combinations of the V_(H)CDRs of these two antibodies. As a specific non-limiting example toillustrate the various combinations possible, such an antibody couldinclude in its V_(L) chain, CDRs 1 and 2 of MAb5 (V_(L) CDR 1.5 andV_(L) CDR 2.5, respectively) and CDR 3 of MAb6 (V_(L) CDR 3.6), and inits V_(H) chain, CDR 1 of MAb6 (V_(H) CDR 1.6) and CDRs 2 and 3 of MAb5(V_(H) CDR 2.5 and V_(H) CDR 3.5, respectively). Amino acid sequences ofCDRs of antibodies (also known as hypervariable regions) produced byhybridomas that have been deposited can be obtained using conventionalmeans.

As is known in the art, the amino acid position/boundary delineating ahypervariable region of an antibody can vary, depending on the contextand the various definitions known in the art. Some positions within avariable domain may be viewed as hybrid hypervariable positions in thatthese positions can be deemed to be within a hypervariable region underone set of criteria while being deemed to be outside a hypervariableregion under a different set of criteria. One or more of these positionscan also be found in extended hypervariable regions. The anti-PGantibodies described herein may contain modifications in these hybridhypervariable positions. The variable domains of native heavy and lightchains each comprise four FR regions, largely by adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions in theorder FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the otherchain, contribute to the formation of the target binding site ofantibodies (see Kabat et al., 1987, Sequences of Proteins ofImmunological Interest, National Institute of Health, Bethesda, Md.). Asused herein, numbering of immunoglobulin amino acid residues is doneaccording to the immunoglobulin amino acid residue numbering system ofKabat et al., unless otherwise indicated.

With reference to TABLE 2A, specific embodiments of N-terminal anti-hPGantibodies useful in the methods and kits described herein include, butare not limited to, the following:

(a) antibodies having V_(L) CDRs that correspond in sequence to theV_(L) CDRs of MAb1, MAb2, MAb3, MAb4, MAb15, MAb16, MAb17, MAb18, MAb19or MAb20, and V_(H) CDRs that correspond in sequence to the V_(H) CDRsof MAb1, MAb2, MAb3, MAb4, MAb15, MAb16, MAb17, MAb18, MAb19 or MAb20;

(b) antibodies having V_(L) CDRs and V_(H) CDRs that correspond insequence to the V_(L) and V_(H) CDRs of MAb1, MAb2, MAb3, MAb4, MAb15,MAb16, MAb17, MAb18, MAb19 or MAb20;

(c) antibodies in which:

-   -   (i) V_(L) CDR 1 is selected from QSIVHSNGNTY (“V_(L) CDR 1.3”;        SEQ ID NO:4), QSLVHSSGVTY (“V_(L) CDR 1.4”; SEQ ID NO:10),        QSLLDSDGKTY (“V_(L) CDR 1.16”; SEQ ID NO:50), and SQHRTYT        (“V_(L) CDR 1.19”; SEQ ID NO:51);    -   (ii) V_(L) CDR 2 is selected from KVS (“V_(L) CDR 2.3” or “V_(L)        CDR 2.4”; SEQ ID NO:5), LVS (“V_(L) CDR 2.16”; SEQ ID NO:53),        and VKKDGSH (“V_(L) CDR 2.19”; SEQ ID NO:54);    -   (iii) V_(L) CDR 3 is selected from FQGSHVPFT (“V_(L) CDR 3.3”;        SEQ ID NO:6), SQSTHVPPT (“V_(I), CDR 3.4”; SEQ ID NO:11),        WQGTHSPYT (“V_(L) CDR 3.16”; SEQ ID NO:57), and GVGDAIKGQSVFV        (“V_(L) CDR 3.19”; SEQ ID NO:58);    -   (iv) V_(H) CDR 1 is selected from GYIFTSYW (“V_(H) CDR 1.3”; SEQ        ID NO:1), GYTFSSSW (“V_(H) CDR 1.4”; SEQ ID NO:7), GYTFTSYY        (“V_(H) CDR 1.16”; SEQ ID NO:39), and GYSITSDYA (“V_(H) CDR        1.19”; SEQ ID NO:40);    -   (v) V_(H) CDR 2 is selected from FYPGNSDS (“V_(H) CDR 2.3”; SEQ        ID NO:2), FLPGSGST (“V_(H) CDR 2.4”; SEQ ID NO:8), INPSNGGT        (“V_(H) CDR 2.16”; SEQ ID NO:43), and ISFSGYT (“V_(H) CDR 2.19”;        SEQ ID NO:44); and    -   (vi) V_(H) CDR 3 is selected from TRRDSPQY (“V_(H) CDR 3.3”; SEQ        ID NO:3), ATDGNYDWFAY (“V_(H) CDR 3.4” SEQ ID NO:9), TRGGYYPFDY        (“V_(H) CDR 3.16”; SEQ ID NO:47), and AREVNYGDSYHFDY (“V_(H) CDR        3.19”; SEQ ID NO:48);

(d) antibodies having a V_(L) that corresponds in sequence to the V_(L)of MAb1, MAb2, MAb3, MAb4, MAb15, MAb16, MAb17, MAb18, MAb19 or MAb20and a V_(H) that corresponds in sequence to the V_(H) of MAb1, MAb2,MAb3, MAb4, MAb15, MAb16, MAb17, MAb18, MAb19 or MAb20; and

(e) antibodies having a V_(L) and a V_(H) that corresponds in sequenceto the V_(L) and V_(H) of MAb1, MAb2, MAb3, MAb4, MAb15, MAb16, MAb17,MAb18, MAb19 or MAb20.

With reference to TABLE 2B, specific embodiments of C-terminal anti-hPGantibodies useful in the methods and kits described herein include, butare not limited to, the following:

(a) antibodies having V_(L) CDRs that correspond in sequence to theV_(L) CDRs of MAb5, MAb6, MAb7, MAb8, MAb9, MAb10, MAb11, MAb12, MAb13,MAb14, MAb21, MAb22 or MAb23 and V_(H) CDRs that correspond in sequenceto the V_(H) CDRs of MAb5, MAb6, MAb7, MAb8, MAb9, MAb10, MAb11, MAb12,MAb13, MAb14, MAb21, MAb22 or MAb23;

(b) antibodies having V_(L) CDRs and V_(H) CDRs that correspond insequence to the V_(L) and V_(H) CDRs of MAb5, MAb6, MAb7, MAb8, MAb9,MAb10, MAb11, MAb12, MAb13, MAb14, MAb21, MAb22 or MAb23;

(c) antibodies in which:

-   -   (i) V_(L) CDR 1 is selected from KSLRHTKGITF (“V_(L) CDR 1.8”;        SEQ ID NO:49) and QSLLDSDGKTY (“V_(L) CDR 1.13”; SEQ ID NO:50);    -   (ii) V_(L) CDR 2 is selected from QMS (“V_(L) CDR 2.8”; SEQ ID        NO:52) and LVS (“V_(L) CDR 2.13”; SEQ ID NO:53);    -   (iii) V_(L) CDR 3 is selected from AQNLELPLT (“V_(L) CDR 3.8”;        SEQ ID NO:55) and WQGTHFPQT (“V_(L) CDR 3.13”; SEQ ID NO:56);    -   (iv) V_(H) CDR 1 is selected from GFTFTTYA (“V_(H) CDR 1.8”; SEQ        ID NO:37) and GFIFSSYG (“V_(H) CDR 1.13”; SEQ ID NO:38);    -   (v) V_(H) CDR 2 is selected from ISSGGTYT (“V_(H) CDR 2.8”; SEQ        ID NO:41) and INTFGDRT (“V_(H) CDR 2.13”; SEQ ID NO:42); and    -   (vi) V_(H) CDR 3 is selected from ATQGNYSLDF (“V_(H) CDR 3.8”;        SEQ ID NO:45) and ARGTGTY (“V_(H) CDR 3.13”; SEQ ID NO:46);

(d) antibodies having a V_(L) that corresponds in sequence to the V_(L)of MAb5, MAb6, MAb7, MAb8, MAb9, MAb10, MAb11, MAb12, MAb13, MAb14,MAb21, MAb22 or MAb23 and a V_(H) that corresponds in sequence to theV_(H) of MAb5, MAb6, MAb7, MAb8, MAb9, MAb10, MAb11, MAb12, MAb13,MAb14, MAb21, MAb22 or MAb23; and

(e) antibodies having a V_(L) and a V_(H) that correspond in sequence tothe V_(L) and V_(H) that correspond in sequence to the V_(L) and V_(H)of MAb5, MAb6, MAb7, MAb8, MAb9, MAb10, MAb11, MAb12, MAb13, MAb14,MAb21, MAb22 or MAb23.

As will be appreciated by skilled artisans, anti-hPG antibodies usefulin the diagnostic methods can be of any origin, including, for example,mammalian (e.g., human, primate, rodent, goat or rabbit), non-mammalian,or chimeric in nature (derived from more than one species of origin).Antibodies suitable for therapeutic uses in animals, including humans,are preferably derived from the same species intended to be treated, orhave been modified or designed to be non-immunogenic or have reducedimmunogenicity in the animal being treated. A specific class of anti-hPGantibodies useful for therapeutic uses in humans is the class ofhumanized antibodies, discussed in more detail, below. Anti-hPGantibodies useful in the methods and kits described herein can also beof, or derived from, any isotype, including, for example, IgA (e.g.,IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4) or IgM.Anti-hPG antibodies designed for therapeutic uses are preferably of theIgG isotype.

In some embodiments, anti-hPG antibodies useful for therapeutic methodsdescribed herein are humanized. In general, humanized antibodiescomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence, and can be referred to as “CDR-grafted.” The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin consensussequence. Methods for humanizing antibodies, including methods fordesigning humanized antibodies, are well-known in the art. See, e.g.,Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77; Lefranc et al.,2009, Nucl. Acids Res. 37:D1006-1012; Lefranc, 2008, Mol. Biotechnol.40: 101-111; Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.5,530,101, 5,585,089, 5,693,761, 5,693,762 and 6,180,370 to Queen etal.; EP239400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539;EP592106; EP519596; Padlan, 1991, Mol. Immunol. 28:489-498; Studnicka etal., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad.Sci. 91:969-973; and U.S. Pat. No. 5,565,332, the disclosures of whichare hereby incorporated by reference in their entireties.

Humanized versions of antibodies having CDR sequences corresponding tothe CDRs of non-human anti-hPG antibodies, including by way of exampleand not limitation, the various N-terminal anti-hPG monoclonalantibodies provided in TABLE 2A and the various C-terminal anti-hPGmonoclonal antibodies provided in TABLE 2B, can be obtained using thesewell-known methods. Projected sequences for humanized V_(L) and V_(H)chains of selected anti-hPG antibodies are provided in TABLES 2A and 2B.Specific examples of humanized antibodies include antibodies comprising:

(a) any three V_(L) CDRs and any three V_(H) CDRs disclosed herein;

(b) a heavy chain variable region comprising an amino acid sequencecorresponding to SEQ ID NO:21 and a light chain variable regioncomprising an amino acid sequence corresponding to SEQ ID NO:22;

(c) a heavy chain variable region comprising an amino acid sequencecorresponding to SEQ ID NO:23 and a light chain variable regioncomprising an amino acid sequence corresponding to SEQ ID NO:24;

(d) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:75, 77, and 79 and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:76 and 78;

(e) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:80 and 82 and a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:81 and 83;

(f) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:84, 86, and 88 and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:85, 87, and 89; and

(g) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:90, 92, and 94 and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:91, 93, and 95.

As will be recognized by skilled artisans, anti-hPG antibodies havingspecific binding properties, such as the ability to bind a specificepitope of interest, can be readily obtained using the various antigensand immunogens described herein and assessing their ability to competefor binding hPG with a reference antibody of interest. Any of theanti-hPG antibodies described herein can be utilized as a referenceantibody in such a competition assay. A specific assay useful forassessing the ability of an antibody to compete for binding hPG with abiotinylated reference anti-hPG antibody of interest is provided inExample 5.

In conducting an antibody competition study between a reference anti-hPGantibody and any test antibody (irrespective of species or isotype), onemay first label the reference with a label detectable either directly,such as, for example, a radioisotope or fluorophore, or indirectly, suchas, for example biotin (detectable via binding withfluorescently-labeled streptavidin) or an enzyme (detectable via anenzymatic reaction), to enable subsequent identification. In this case,a labeled reference anti-hPG antibody (in fixed or increasingconcentrations) is incubated with a known amount of hPG, forming anhPG:labeled anti-hPG antibody complex. The unlabeled test antibody isthen added to the complex. The intensity of the complexed label ismeasured. If the test antibody competes with the labeled referenceanti-hPG antibody for hPG by binding to an overlapping epitope, theintensity of the complexed label will be decrease relative to a controlexperiment carried out in the absence of test antibody.

Numerous methods for carrying out binding competition assays are knownand can be adapted to yield results comparable to the assay describedabove and in Example 5.

An antibody is considered to compete for binding hPG with a referenceanti-hPG antibody, and thus considered to bind approximately the same oran overlapping epitope of hPG as the reference anti-hPG antibody, if itreduces binding of the reference anti-hPG antibody to hPG in acompetitive binding assay, and specifically the competitive bindingassay of Example 5, by at least 50%, at a test antibody concentration inthe range of 0.01-100 μg/mL (e.g., 0.01 μg/mL, 0.08 μg/mL, 0.4 μg/mL, 2μg/mL, 10 μg/mL, 50 μg/mL or 100 μg/mL or other concentration within thestated range), although higher levels of reduction, for example, 60%,70%, 80%, 90% or even 100%, may be desirable.

Skilled artisans will appreciate that is some contexts, for example,diagnostic and monitoring contexts, it may be desirable to label theanti-PG antibodies. Such labels are useful for detection andquantification. Suitable labels are well known in the art, and can be“direct” in that they are directly observable or detectable (forexample, fluorophores or radioisotopes) or “indirect” in that theyinteract with something else that produces and observable or detectablesignal (for example, an enzyme that acts on a substrate to produce adetectable signal, or a binding molecule such as biotin that binds alabeled, streptavidin molecule). Numerous labeling systems, as well asmeans for labeling antibodies with them, are known in the art, and arecontemplated for use herein.

Although the various anti-hPG antibodies useful in the methods describedherein have been exemplified with full length antibodies, skilledartisans will appreciate that binding fragments, or surrogate antibodiesdesigned or derived from full-length antibodies or binding fragments,may also be used. Suitable fragments, surrogates, etc., include, but arenot limited to, Fab′, F(ab′)2, Fab, Fv, vIgG, scFv fragments andsurrobodies. Unless specified otherwise, the term “antibody” as usedherein is intended to include all forms of antibodies and“antibody-like” surrogate molecules, including single chain antibodies,surrobodies and binding fragments. Antibodies having structures typicalof naturally occurring antibodies are referred to herein as “nativeantibodies.”

7.7 Methods of Producing Anti-PG Antibodies

Anti-PG antibodies useful in the methods described herein may beobtained using standard, well-known methods. To express anti-PGantibodies useful in the methods described herein, DNAs encoding partialor full-length light and heavy chains are inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevectors or, more typically, both genes are inserted into the sameexpression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the anti-PGantibody light or heavy chain sequences, the expression vector canalready carry antibody constant region sequences. For example, oneapproach to converting the anti-PG antibody V_(H) and V_(L) sequences tofull-length antibody genes is to insert them into expression vectorsalready encoding heavy chain constant and light chain constant regions,respectively, such that the V_(H) segment is operatively linked to theC_(H) segment(s) within the vector and the V_(L) segment is operativelylinked to the C_(L) segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif., 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors can carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, puromycin,blasticidin, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in DHFR⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE-dextran transfection and the like.

It is possible to express the antibodies described herein in eitherprokaryotic or eukaryotic host cells. In certain embodiments, expressionof antibodies is performed in eukaryotic cells, e.g., mammalian hostcells, for optimal secretion of a properly folded and immunologicallyactive antibody. Exemplary mammalian host cells for expressing therecombinant antibodies of the disclosure include Chinese Hamster Ovary(CHO cells) (including DHFR⁻ CHO cells, described in Urlaub & Chasin,1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman & Sharp, 1982, Mol.Biol. 159:601-621), NS0 myeloma cells, COS cells, 293 cells and SP2/0cells. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods. Host cells can also be used to produceportions of intact antibodies, such as F_(ab) fragments or scF_(v)molecules. It is understood that variations on the above procedure arewithin the scope of the present disclosure. For example, it can bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an anti-PG antibody describedherein.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to PG. The molecules expressed from such truncatedDNA molecules are also useful in the methods described herein.

For recombinant expression of an anti-PG antibody, the host cell can beco-transfected with two expression vectors, the first vector encoding aheavy chain derived polypeptide and the second vector encoding a lightchain derived polypeptide. Typically, the two vectors each contain aseparate selectable marker. Alternatively, a single vector can be usedwhich encodes both heavy and light chain polypeptides.

Anti-PG antibodies can also be produced by chemical synthesis (e.g., bythe methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also begenerated using a cell-free platform (see, e.g., Chu et al., 2001,Biochemia No. 2 (Roche Molecular Biologicals)).

Once an anti-PG antibody has been produced by recombinant expression orsynthetic means, it can be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor PG after Protein A or Protein G selection, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-PG antibodies or binding fragments thereof can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

8. EXAMPLES 8.1 Example 1 Quantification of Plasma or Serum PG Levels

Plasma and/or serum levels of PG can be conveniently determined usingthe following assay. 96-well microtiter plates are coated with between0.5 and 10 μg/mL of a C-terminal anti-hPG antibody, for example, arabbit C-terminal anti-hPG polyclonal antibody, or a C-terminal anti-hPGantibody described herein, and then incubated overnight. Plates are thenwashed three times in PBS-Tween (0.05%) and blocked with 2% (w/v) nonfatdried milk in PBS-Tween (0.05%). Separately, test samples, controlsamples (blank or PG-negative plasma or serum samples), and betweenabout 5 pM (0.5×10⁻¹¹ M) and about 0.1 nM (1×10¹⁰ M) of an hPG referencestandard (lyophilized hPG diluted in PG-negative plasma or serum) areprepared in an appropriate diluent (e.g., PBS-Tween 0.05%). Samples areincubated on the coated plates for between 2 and 4 hours at 37° C., oralternatively between 12 and 16 hours at 21° C. After incubation, platesare washed three times with PBS-Tween (0.05%) and incubated with between0.001 and 0.1 μg/mL of an N-terminal anti-hPG antibody, for example, apolyclonal N-terminal anti-hPG antibody or an N-terminal monoclonalanti-hPG antibody as described herein, coupled to horseradish peroxidase(HRP) (see, Nakane et al., 1974, J. Histochem. Cytochem.22(12):1084-1091) for 30 minutes at 21° C. Plates are then washed threetimes in PBS-Tween (0.05%) and HRP substrate is added for 15 minutes at21° C. The reaction is stopped by added 100 μL of 0.5M sulfuric acid andan optical density measurement is taken at 405 nm. Test sample hPGlevels are determined by comparison to a standard curve constructed fromthe measurements derived from the hPG reference standard.

8.2 Example 2 ELISA Assay for Assessing Specificity of Anti-hPGAntibodies

Specificity of anti-hPG antibodies can be conveniently determined usingan ELISA assays as follows. 96-well plates are incubated overnight at 4°C. with appropriate concentration(s) of test polypeptide (e.g., 25 and50 ng recombinant human PG, and 50 and 250 ng CTFP or othergastrin-derived gene products) in Phosphate-Buffered Saline (PBS), afterwhich the wells are washed three times with wash solution (PBS and 0.1%Tween-20), and then incubated for 2 hours at 22° C. with 100 μL blockingsolution (PBS, 0.1% Tween-20, 0.1% Bovine Serum Albumin or caseinhydrolysate) per well. After blocking, the wells are washed three timesand the antibody to be assayed (test antibody) is added. 100 μL of thetest antibody (at 0.3 to 1 ng/mL) in PBS and 0.1% Tween-20 are added toeach well. Plates are then incubated for 2 hours at 22° C., after whichthe test antibody solution is discarded and replaced, after a wash step(3×100 μL wash solution, as noted above), with blocking solutioncontaining a secondary antibody, a goat anti-mouse IgG (Fc) antibodycoupled to horseradish peroxidase. After a 1-hour incubation withsecondary antibody, 100 μL of substrate solution (e.g. Fast OPD, orO-Phenylenediamine dihydrochloride, available from Sigma-Aldrich Co.,prepared according to manufacturer's directions) is added to each welland incubated in the dark for 20 minutes at 22° C. The reaction isstopped by adding 50 μL of 4N sulfuric acid and the amount of substratecatalyzed determined by measuring the optical density (O.D.) at 492 nm.Substrate conversion is proportional to the amount of primary (test)antibody bound to the antigen. Experiments are run in duplicate and ODmeasurements plotted as a function of antigen concentration. Testantibodies are scored as specific for PG if the measured O.D. is between0.2 and 1.5 for hPG and there is no statistically significant signalabove background with CTFP or any of the other gastrin-gene derivedpeptides, where the background is the average signal from control wellscontaining only PBS.

8.3 Example 3 Assay for Assessing Neutralizing Activity of Anti-hPGAntibodies

A specific test for assessing whether a specific anti-hPG antibody isneutralizing can be performed as follows. LS 174T cells are seeded in 6wells of a 6-well plate, at approximately 50,000 cells per well. Cellsare then treated at 12-hour intervals for 48 hours with the testanti-hPG antibody or a control antibody, at antibody concentrations ofabout 5 μg/mL. A test antibody is defined as neutralizing in the assay,if the number of cells treated with the test antibody shows astatistically significant reduction of at least 10% in the number ofsurviving cells compared to the number of cells treated with a control,non-specific antibody, using a two-tailed Mann-Whitney test (withdifferences considered as significant when p<0.05). Total cell numbersare corrected for the number of cells at the start of the treatmentperiod, referred to as T₀.

8.4 Example 4 Assay for Assessing Affinity of an Anti-hPG Antibody

Affinity constants of anti-hPG antibodies can be measured using theProteon Technique (BioRad), according to Nahshol et al., 2008,Analytical Biochemistry 383:52-60, hereby incorporated by reference inits entirety. Briefly, for murine anti-PG antibodies, an anti-mouse IgGantibody (50 μg/ml) is first coated on a sensor chip, making sure thatthe signal detected by the chip after injection of the antibody fallsbetween 10,000 and 11,500 response units (RU). The murine anti-hPGantibody of interest (test antibody) is then injected (at a typicalconcentration of 30 μg/ml). If the test antibody binds sufficiently, andadditional signal of at least 500 RU will be observed. A time-course ofbinding between test antibody and hPG is then obtained by injectingvarying concentrations of hPG, for example 200 nM, 100 nM, 50 nM, 25 nM,and 12.5 nM, and detecting the level of association. Typically, severalchannels are available to test multiple antibodies in parallel in asingle experiment, making it possible to assay binding of a single testantibody at different concentrations of hPG in parallel. One channelshould be injected with a murine monoclonal antibody that is notspecific to hPG as a control for non-specific binding and anotherchannel should be injected with dilution buffer alone as a baseline forthe background signal. Generally, no binding is detectable in thechannel injected with non-specific murine antibody. Antibodiesdisplaying a high level of association in this setting, which may resultin saturation of the trapped monoclonal antibody by hPG, can be testedagainst lower hPG concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM and3.125 nM), allowing for a more refined measurement.

Affinity constants (K_(D)) are calculated as the ratio between thedissociation constant (k_(d)) and the association constant (k_(a)).Experimental values can be validated by analyzing the statisticallyrelevant similarity between experimental curves based on bindingmeasurements and theoretical profiles.

Affinity constants of non-murine anti-hPG antibodies can be assessed ina similar format using an IgG specific for the species of origin of theanti-hPG test antibody.

8.5 Example 5 Assay for Assessing Competitive Binding with a ReferenceAnti-hPG Antibody

A specific assay for assessing whether an antibody of interest (testantibody) competes for binding hPG with a biotinylated referenceanti-hPG antibody can be performed as follows. 96-well plates are coatedwith a capture anti-hPG antibody (polyclonal or monoclonal antibodyrecognizing an N- or C-terminal region of hPG that differs from theepitope recognized by the biotinylated reference anti-hPG antibody), ata concentration to be chosen within the range of 1-10 μg/ml, overnightat 4° C. (0.1 to 1 μg/well). After blocking with blocking buffer (0.1%Tween-20, 0.1% BSA in PBS) for 2 hr at 22° C., recombinant hPG is addedat a concentration ranging between 10 pM to 1 nM (10 to 1000 pg/well)and incubated for 2 hr at 22° C. Thereafter, the biotinylated referenceanti-hPG antibody (or a mixture containing the biotinylated referenceanti-hPG antibody) is added, along with increasing concentrations ofunlabeled test antibody, and incubated for 1 hr at 22° C. After washingto remove unbound antibodies, detection of bound labeled referenceanti-hPG antibody is performed by incubating the mixture with 50 ng/mlstreptavidin-HRP for 1 hr at 22° C., followed by incubation with achemiluminescent substrate for horseradish peroxidase for 5 minutes at22° C., and then quantifying the relative light units (RLU) in aluminometer. Assays are performed in duplicate.

Antibodies that compete with a reference anti-hPG antibody inhibit thebinding of the reference antibody to hPG. An antibody that binds tosubstantially the same epitope, or with an overlapping epitope, as thereference antibody significantly reduces (for example, by at least 50%)the amount of reference anti-hPG antibody bound, as evidenced by areduction observed RLUs.

A high control value is obtained from a control experiment carried outby incubating the labeled reference antibody with recombinant hPGwithout test antibody. A low control value is obtained from a controlexperiment carried out by incubating the labeled reference antibody withrecombinant hPG in the presence of excess concentrations of theunlabeled reference antibody (the unlabeled reference antibody thuscompeting with the labeled antibody for binding to hPG). The capacity oftest antibodies to compete with the reference anti-hPG antibody is thendetermined by incubating the labeled reference antibody with recombinanthPG in the presence of increasing concentrations of the unlabeled testantibody.

In a test assay, a significant reduction in the observed RLUs in thepresence of a test antibody indicates that the test antibody recognizessubstantially the same epitope as the reference anti-hPG antibody.

The inhibition of binding can be expressed as an inhibition constant, orwhich is calculated according to the following formula:

K_(i)=IC₅₀/[1+(reference anti-hPG Ab concentration/K_(D)^(reference anti-hPG Ab))]

where “IC₅₀” is the concentration of test antibody that yields a 50%reduction in binding of the reference antibody and K_(D)^(reference anti-hPG Ab) is the dissociation constant of the referenceanti-hPG antibody, a measure of its affinity for hPG. Useful testantibodies that compete with a reference anti-hPG antibody (for example,one of the anti-hPG antibodies described herein) will typically haveK_(i)s ranging from 10 pM to 100 nM under assay conditions describedherein.

8.6 Example 6 Detection of Serum PG in Samples from Patients withFamilial Adenomatous Polyposis

This example shows that elevated serum PG levels can be correlated withthe presence of polyps in individual with FAP.

8.6.1 Methods

Serum PG levels were quantified as described in Example 1 in samplesfrom 6 patients with Familial Adenomatous Polyposis. Serum samples wereobtained from patients with the following characteristics:

-   -   Two individuals (A and B), both older than 55, having previously        undergone colectomy, regularly monitored by endoscopy, in whom        no polyps have been detected since surgery.    -   One individual (C), aged 30, having previously undergone        colectomy and follow up surgery to remove further polyps. The        individual had surgery several months before the blood sample        was collected.    -   One individual (D), aged 27, having previously undergone        colectomy, presenting with multiple polyps in the small        intestine (but no cancer) at the time the blood sample was        collected.    -   One individual (E), aged 52, having previously undergone        colectomy presenting with multiple polyps in the rectum at the        time a blood sample was collected.    -   One individual (F), aged 10, presenting with multiple colorectal        polyps at the time a blood sample was collected.

8.6.2 Results

Results shown Table 5 below are expressed as mean PGconcentration±standard deviation (pM):

TABLE 5 Mean PG Concentration Patient (pM) ±s.d. A  6.9 ± 3.3 B 0.0 C0.0 D  167.5 ± 43.0 E 351.85 ± 96.0 F   233 ± 11.3

Results indicate that PG levels are particularly elevated in individualsbearing a high number of polyps at the time of sampling. In comparison,patients who have undergone surgery display very low or undetectablelevels of PG.

8.7 Example 7 Detection of Serum PG in Samples from Patients withPre-Cancerous Sporadic Adenomatous Polyps

This example demonstrates that more than twent-five percent ofindividuals with sporadic adenomatous polyposis have increased serum PGlevels.

8.7.1 Methods

PG levels were measured in two different sample sets: a first set ofsamples obtained from twenty-five individuals having multipleadenomatous polyps, similar to the number that would be found insubjects with FAP, and a second set of samples from a plasma bank,collected from 104 individuals ranging in age from 45 to 65 years.Plasma progastrin levels were quantified using an ELISA assay, asdescribed above in Example 1.

8.7.2 Results

23% (12/52) of subjects with adenomatous polyps studied had progastrinlevels above 50 pM. By comparison, 16.3% (17/104) of subjects in fromthe blood bank group had PG levels above 50 pM. The medical history ofthe individuals whose samples were banked is unknown. Healthyindividuals have low levels of PG, not typically exceeding 50 pM. See,e.g., Siddheshwar et al., 2001, “Plasma levels of progastrin but notamidated gastrin or glycine extended gastrin are elevated in patientswith colorectal carcinoma,” Gut 48:47-52. Levels above 50 pM are thoughtto be indicative of an underlying pathology.

Almost one quarter of the individuals in whom polyps were found also hadelevated PG levels. This is consistent with the observation that about20% of sporadic adenomatous polyps develop into malignant tumors, atransformation that is thought to be accompanied by elevated PG levels.Therefore, elevated PG levels in the presence of adenomatous polyps canserve as a useful metric in identifying patients for further follow-upor for prophylactic anti-PG treatment.

With respect to the samples from the plasma bank, it is likely that someor all of the banked samples with PG levels above 50 pM came fromindividuals with underlying conditions that caused elevated PG levels.Use of appropriately screened control samples would likely show agreater difference between the percentage of individuals with sporadicadenomatous polyps and those without polyps who have PG levels above 50pM.

8.8 Example 8 Anti-PG Compositions Prevent Tumor Development in a MouseModel of FAP

This example demonstrates the ability of anti-hPG antibodies to preventthe formation of tumors in vivo.

8.8.1 Methods

Transgenic mice carrying a mutation in an allele of the AdenomatousPolyposis Coli (APC) gene similar to that found in individuals withFamilial Adenomatous Polyposis (FAP), were treated with an anti-hPGantibody. These mice, referred to as APCΔ14 mice, spontaneously developtumors in their intestines when the second (wild type) APC allele islost via a “loss of heterozygocity” (LOH) mechanism, (Colnot et al.,2004, “Colorectal cancers in a new mouse model of familial adenomatouspolyposis: influence of genetic and environmental modifiers,” LabInvestigation 84:1619-1630). The first detectable tumors can be foundaround 2 months of age, and by 3.5 months, the number of tumors isgenerally around 15-20. These tumors have been shown to produceprogastrin.

Four-month-old APCΔ14 mice were treated twice a week for six weeks witheither a control polyclonal antibody antibody—a rabbit anti-human IgGantiserum (Jackson ImmunoResearch (reference no. 309-005-0089)—or ananti-PG polyclonal antibody, raised against (1) an N-terminal peptideand (2) a C-terminal peptide as described in Hollande et al., WO07/135,542, by intra-peritoneal injection at a dose of 9 mg/kg. The micewere weighed once a week. At the end of the six-week treatment regimen,their intestines were photographed, the total number of tumors counted.There were six mice in the treatment and the control group. Genotypingidentified two mice from the control group that did not carry theexpected heterozygous APC mutation, and were excluded from theexperiment.

8.8.2 Results

Results are shown below in Table 6. Mice treated with control antibodyexhibited a total of 125 tumors, with 31.25 tumors on average per mouse.Anti-PG treated mice has 46 total tumors or, on average, 7.6 tumors permouse. This difference is statistically significant (Mann-Whitney test,P=0.0095).

TABLE 6 Treatment (no. of mice) Number of tumors per mouse Control PAb(4) 23 48 28 26 Anti-hPG PAb (6) 2 16 15 9 2 2

Results indicate that the number of tumors found in four out the sixanimals treated with anti-progastrin antibodies falls below the averagenumber of fifteen to twenty tumors generally found in APCΔ14 mice at 3.5months of age. See, Table 6 below. These data indicate that treatmentwith anti-progastrin antibodies prevents new tumors from developing inthese animals.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A method of preventing gastrointestinal cancer ina human subject, comprising ministering to a human subject predisposedto develop adenomatous polyps an amount of an anti-PG antibodysufficient to provide a therapeutic benefit.
 2. The method of claim 1,in which the anti-PG antibody is an anti-hPG monoclonal antibody.
 3. Themethod of claim 2 in which the anti-hPG monoclonal antibody is ahumanized anti-hPG monoclonal antibody.
 4. The method of claim 2 inwhich the anti-hPG monoclonal antibody is an N-terminal anti-hPGmonoclonal antibody.
 5. The method of claim 4 in which the N-terminalanti-hPG monoclonal antibody binds an epitope comprising a sequenceselected from the group consisting of DAPLG (SEQ ID NO:28), PDAPLG (SEQID NO:29), PRSQQPD (SEQ ID NO:30), WKPRSQQPD (SEQ ID NO:31) andWKPRSQQPDAPLG (SEQ ID NO:32).
 6. The method of claim 4 in which theN-terminal anti-hPG monoclonal antibody is raised against an immunogencomprising a peptide having the sequence SWKPRSQQPDAPLG (SEQ ID NO:25).7. The method of claim 4 in which the N-terminal anti-hPG monoclonalantibody comprises three V_(H) CDRs corresponding in sequence to theV_(H) CDRs of MAb3, MAb4, MAb15, MAb16 or MAb19 and three V_(L) CDRscorresponding in sequence to the V_(L) CDRs of MAb3, Mab4, MAb15, MAb16or MAb19.
 8. The method of claim 4 in which the N-terminal anti-hPGmonoclonal antibody comprises CDRs corresponding in sequence to the CDRsof MAb3, MAb4, MAb15, MAb16 or MAb
 19. 9. The method of claim 4 in whichthe anti-hPG monoclonal antibody competes for binding hPG with areference antibody selected from the group consisting of MAb3, MAb4,MAb15, MAb16 and MAb19.
 10. The method of claim 2 in which the anti-hPGmonoclonal antibody is a C-terminal anti-hPG monoclonal antibody. 11.The method of claim 10 in which the C-terminal anti-hPG monoclonalantibody binds an epitope comprising a sequence selected from the groupconsisting of FGRR (SEQ ID NO:33), MDFGR (SEQ ID NO:34), AEDEN (SEQ IDNO:35) and GWMDFGRR (SEQ ID NO:36).
 12. The method of claim 10 in whichthe C-terminal anti hPG monoclonal antibody is raised against animmunogen comprising a peptide having the sequenceQGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO:27).
 13. The method of claim 10 inwhich the C-terminal anti-hPG monoclonal antibody comprises three V_(H)CDRs corresponding in sequence to the CDRs of MAb5, MAb6, MAb7, MAb8,MAb11, MAb12 or MAb13 and three V_(L) CDRs corresponding in sequence tothe CDRs of MAb5, MAb6, MAb7, MAb8, MAb11, MAb12 or MAb13.
 14. Themethod of claim 10 in which the C-terminal anti hPG monoclonal antibodycomprises CDRs corresponding in sequence to the CDRs of MAb5, MAb6,MAb7, MAb8, MAb11, MAb12 or MAb13.
 15. The method of claim 10 in whichthe C-terminal anti-hPG monoclonal antibody competes for binding hPGwith a reference antibody selected from the group consisting of MAb5,MAb6, MAb7, MAb8, MAb11, MAb12 and MAb13.
 16. The method of claim 2,wherein the subject has a mutation in the APC gene associated withadenomatous polyposis.
 17. The method of claim 16, wherein the subjecthas familial adenomatous polyposis.
 18. The method of claim 16, whereinthe subject has sporadic adenomatous polyposis.
 19. The method of claim2, in which the anti-hPG monoclonal antibody is administered adjunctiveto surgical resection of tissue comprising adenomatous polyps.
 20. Themethod of claim 2, in which the anti-hPG monoclonal antibody isadministered adjunctive to chemotherapy.
 21. The method of claim 2, inwhich the anti-hPG monoclonal antibody is administered adjunctive totreatment with a non-steroidal anti-inflammatory drug.
 22. A method ofidentifying a subject in need of colonoscopy comprising determiningwhether a subject predisposed to develop adenomatous polyps has a plasmaor serum progastrin concentration at or above about 50 pM, where such aconcentration in said subject is indicative of a need for follow-upcolonoscopy.
 23. A method of identifying a subject with increasedprobability of having adenomatous polyps comprising determining thelevel of PG in samples of a body fluid from a subject predisposed todevelop adenomatous polyps, and comparing the level of PG in the sampleto a baseline, wherein a level of PG in the sample above the baselinelevel is indicative of a need for anti-progastrin treatment.
 24. Themethod of claim 23, wherein said baseline is the level of PG in a sampleobtained at an earlier time from the subject.
 25. A method of monitoringthe effectiveness of anti-PG treatment comprising determining whether asubject with adenomatous polyposis has a plasma or serum progastrinconcentration below about 10 pM, where such a concentration indicatesthe treatment is effective.